Hydrogels with biodegradable crosslinking

ABSTRACT

Hydrogels that degrade under appropriate conditions of pH and temperature by virtue of crosslinking compounds that cleave through an elimination reaction are described. The hydrogels may be used for delivery of various agents, such as pharmaceuticals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/486,215, filed on 12 Apr. 2017, which is a continuation of U.S.patent application Ser. No. 14/343,819, having an international filingdate of 7 Sep. 2012, which is the national phase of PCT applicationPCT/US2012/054278 having an international filing date of 7 Sep. 2012,which claims benefit of U.S. Application Ser. No. 61/531,990 filed 7Sep. 2011. The contents of the above patent applications areincorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 670572000611SeqList.txt,date recorded: Aug. 29, 2019, size: 1,072 bytes).

BACKGROUND ART

A hydrogel is a 3-dimensional network of natural or synthetichydrophilic polymer chains in which water (up to 99%) is the dispersionmedium. Fragile macromolecules often require a well-hydrated environmentfor activity and structural integrity, and the high degree of hydrationof a hydrogel may preserve the folding of a protein needed for itsbioactivity. The high water content of the hydrogels render the materialbiocompatible and minimize inflammatory reactions of tissues in contactwith the gel, and provide a flexibility comparable to that of livingtissue. Hydrogels are thus of interest in biomedical engineering, asabsorbent materials for wound dressings and disposable diapers, and ascarriers for extended drug release. Hydrogels have been prepared byphysical or chemical crosslinking of hydrophilic natural or syntheticpolymers.

Examples of hydrogels formed from crosslinking of natural polymersinclude those formed from hyaluronans, chitosans, collagen, dextran,pectin, polylysine, gelatin or agarose (see: Hennink, W. E., et al.,Adv. Drug Del. Rev. (2002) 54:13-36; Hoffman, A. S., Adv. Drug Del. Rev.(2002) 43:3-12). These hydrogels consist of high-molecular weightpolysaccharide or polypeptide chains. Some examples of their use includethe encapsulation of recombinant human interleukin-2 in chemicallycrosslinked dextran-based hydrogels (Cadee, J. A., et al., J Control.Release (2002) 78:1-13) and insulin in an ionically crosslinkedchitosan/hyaluronan complex (Surini, S., et al., J. Control. Release(2003) 90:291-301).

Examples of hydrogels formed by chemical or physical crosslinking ofsynthetic polymers include poly(lactic-co-glycolic)acid (PLGA) polymers,(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate,poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO), PEO-PPO-PEOcopolymers (Pluronic®), poly(phosphazene), poly(methacrylates),poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethyleneimine), and others (see, for example, Hoffman, A. S., Adv. Drug Del. Rev(2002) 43:3-12). Examples of protein-polymer encapsulation using suchhydrogels include the encapsulation of insulin in physically crosslinkedPEG-g-PLGA and PLGA-g-PEG copolymers (Jeong, B., et al.,Biomacromolecules (2002) 3:865-868) and bovine serum albumin inchemically crosslinked acrylate-PGA-PEO-PGA-acrylate macromonomers(Sawhney A. S., et al., Macromolecules (1993) 26:581-587).

Depending on the pore size, degradation of a hydrogel is typicallyrequired for release of the encapsulated compounds. Degradationincreases the size of the pores to the extent that the drug may diffuseout of the interior of the hydrogel into surrounding body fluids.Degradation is further desirable in order to remove the hydrogel fromthe body once drug delivery is complete, as surgical removal of thespent hydrogel carrier is often painful. While many of the knownhydrogels are theoretically biodegradable, in practice the degradationis uncontrolled and thus unpredictable. Thus, a need exists for newhydrogel materials that biodegrade at a predetermined rate.

In order to effect degradation of the hydrogel, it is helpful to havecrosslinking agents that are cleavable under physiological conditions.In one approach, enzymatic cleavage of the crosslinker as a substratecan effect this result. However, dependence on enzymatic degradationresults in inter-patient variability as well as differences between invivo and in vitro results.

The present invention takes advantage of a cleavage mechanism describedin a different context—namely drug release from macromolecular carrierswhich is disclosed, for example in U.S. application US2006/0171920 andin WO2009/158668, WO2011/140393, WO2011/140392 and WO2011/140376. Theelimination reaction relies on a modulating group to control the acidityof a proton; ionization of this proton results in release of the drug.

To applicants' knowledge, this mechanism has not been used to establisha cleavable crosslinker for hydrogels which results in the degradationof the gel.

DISCLOSURE OF THE INVENTION

This invention provides hydrogels that degrade to smaller, solublecomponents in a non-enzymatic process upon exposure to physiologicalconditions and to methods to prepare them. The hydrogels are preparedfrom crosslinking agents that undergo elimination reactions underphysiological conditions, thus cleaving the crosslinking agent from thebackbone of the hydrogel. The invention also relates to the crosslinkingagents themselves and intermediates in forming the hydrogels of theinvention. The biodegradable hydrogels prepared according to the methodsof the invention may be of use in diverse fields, including biomedicalengineering, absorbent materials, and as carriers for drug delivery.

Thus, in one aspect, the invention is directed to a hydrogel that isbiodegradable under physiological conditions which hydrogel comprisesone or more polymers crosslinked by a linker that decomposes by anelimination reaction. More specifically, the hydrogels contain linkersthat when disposed in the polymer residues of formula (1):

wherein at least one of R¹, R² or R⁵ along with X is coupled to said oneor more polymers.

-   -   Alternatively, the linker is a residue of formula (2):

wherein at least two of said R¹, R² or R⁵ are coupled to one or morepolymers.

The definitions of R¹, R², R⁵, m, X, W, s, n, t, and Q are set forth indetail herein-below. In the case of formula (2), the coupling may bethrough two R¹'s that exist in the same molecule of formula (2) orthrough one R¹ and one R⁵, for example, in formula (2). That is therequirement that at least two of these substituents as coupled to one ormore polymers simply means that in the crosslinker of formula (2)itself, there must be at least two points of attachment. In someembodiments the R¹, R² and R⁵ substituents are uniform in each of the t“arms”.

The hydrogel may further contain one or more drugs. The drug(s) may besimply contained in the pores of the hydrogel, or may be coupled to acrosslinking agent which is in turn coupled to the polymeric backbone ofthe hydrogel.

The invention also provides methods for preparing biodegradablehydrogels comprising either simultaneously or sequentially contacting atleast one reactive polymer and a cleavable crosslinker compound whereinsaid cleavable crosslinker compound comprises a functional group thatreacts with the reactive polymer and a moiety that cleaves byelimination under physiological conditions also comprising a functionalgroup that reacts with one or more polymers. The invention also providesmethods for the preparation of drug-releasing biodegradable hydrogelswherein the rates of drug release and of hydrogel biodegradation arecontrolled.

Thus, the drugs or other agent may simply be entrapped in the hydrogelor may be included in the hydrogel by virtue of coupling through alinker that releases the drug through an elimination reaction as well,without necessity for the degradation of the gel itself.

In another aspect, the invention provides crosslinking reagentscomprising a moiety capable of being cleaved by elimination underphysiological conditions and further comprising reactive groups capableof forming covalent bonds with reactive polymers.

In still another aspect, the invention provides intermediates formed byreaction of the crosslinking reagents of the invention, with at leastone reactive polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of the invention wherein hydrogels areformed by crosslinking a multi-arm polymer with a crosslinker of formula(1). A 4-arm polymer wherein each arm is terminated with a cyclooctyne(CO) and a crosslinker of formula (1) wherein one R⁵ is (CH₂)_(r)N₃ andX is O—CO—NH—CH₂CH₂(OCH₂CH₂)_(p)—N₃ (Example 20) provides a 4×4 hydrogelcomprising a beta-eliminative linker L in each crosslink. Thedegradation rate of the hydrogel is controlled by appropriate choice ofthe modulating group R¹ on linker L. Also illustrated is the formationof (1) by reaction of a succinimidyl carbonate with an amino-PEG-azide.

FIGS. 2A and 2B illustrate two embodiments of the invention whereinhydrogels are formed by crosslinking multi-arm polymers with compoundsof formula (2). FIG. 2A shows crosslinking a 4-arm polymer wherein eacharm is terminated with a cyclooctyne (CO) with another 4-arm polymer offormula (2) wherein each arm is terminated with a beta-eliminativelinker azide (L₂-N₃). The resulting 4×4 hydrogel comprises abeta-eliminative linker in each crosslink. The degradation rate of thehydrogel is controlled by appropriate choice of the linker L₂. FIG. 2Bshows crosslinking an 8-arm polymer wherein 4 arms are terminated with acyclooctyne (CO) and the remaining arms are attached to either anerosion probe (EP) or a releasably-linked drug (L₁-D). Crosslinking witha 4-arm polymer wherein each arm is terminated with a beta-eliminativelinker azide (L₂-N₃) provides a 4×8 hydrogel comprising abeta-eliminative linker L₂ in each crosslink and comprising drug Dcovalently attached through another beta-eliminative linker L₁. Therates of drug release from the hydrogel and hydrogel degradation arecontrolled by appropriate choices of the linkers L₁ and L₂,respectively.

FIG. 3 shows degradation of 4×4 PEG hydrogels at pH 7.4, 37° C. asmeasured by solubilized fluorescein-PEG fragments described in Example28; reverse gelation times using different modulators:R¹=(4-chlorophenyl)SO₂, 30 hrs, R¹=phenyl-SO₂, 55 hrs;R¹═O(CH₂CH₂)₂NSO₂, 22 days; R¹═CN, 105 days. Solubilized fluorescein wasused as erosion probe, with degelation times being defined as the pointof complete dissolution.

FIG. 4 shows the correlation between the degelation times measured for4×4 hydrogels of Example 28 and the rate of5-(aminoacetamido)fluorescein release measured from soluble PEGconjugates using equivalent linkers.

FIG. 5 shows the pH dependence for degelation of 4×4 PEG hydrogels ofExample 28, wherein L₂ has modulator R¹=(4-chlorophenyl)SO₂.

FIG. 6 shows the correlation between pH and degelation time for the gelsof Example 28.

FIG. 7 shows the release of drug surrogate 5-(aminoacetamido)fluoresceinfrom 4×8 PEG hydrogels of Example 29.

FIG. 8 shows the pH dependence of the release of drug surrogate5-(aminoacetamido)fluorescein from 4×8 PEG hydrogels of Example 29. Thehalf-lives for release were measured at pH 7.4 (23.0 h); pH 7.8 (14.0h); pH 8.1 (6.9 h); pH 8.4 (3.2 h); pH 8.7 (1.9 h); and pH 9.0 (1.1 h).

FIG. 9 shows the correlation between the pH and the half-lives for drugrelease from the 8×4 hydrogels of Example 29.

FIG. 10 shows the release of the peptide exenatide (exendin-4)covalently attached via a releasable linker L₁ having modulatorR¹¹═CH₃SO₂ to an 8×4 PEG hydrogel crosslinked with degradable linkers L₂having modulator R¹═CN at pH 8.8, 37° C. (Example 33). Knowing thepH-dependence of linker release and gel degradation, the correspondingscale at pH 7.4 is also given. Total solubilized exenatide (circles) isreleased with apparent t_(1/2)=20.7 h at pH 8.8, corresponding tot_(1/2)=21 days at pH 7.4. Degelation (squares=solubilized fluoresceinerosion probe) was observed at 172 h at pH 8.8, corresponding to 180days at pH 7.4.

FIG. 11 illustrates in one embodiment the drug-releasing hydrogels areformed by reaction of a first polymer comprising at least two orthogonalfunctional groups (B and C) is reacted with a linker-drug of formula (3)wherein the linker-drug comprises a functional group (B′) that reactswith only one of the orthogonal functional groups (B) present on thefirst polymer, connecting the linker-drug to the first polymer viaresidue B*. The remaining orthogonal functional group (C) on theresulting drug-loaded first polymer (is used to form a hydrogel byreaction with a compound of formula (1) or (2) wherein these compoundscomprise a functional group (C′) that reacts with only the remainingorthogonal functional group present on the drug-loaded first polymer tocrosslink the hydrogel via residue C*.

MODES OF CARRYING OUT THE INVENTION

The hydrogels of the invention are polymer(s) crosslinked by linkersthat decouple the polymer(s) by “elimination.” “Elimination” is areaction mechanism by which a proton H and a leaving group X are removedfrom a molecule so as to form an alkene. In one embodiment of theinvention, the elimination is a 1,2-elimination illustrated as

In other embodiments of the invention, the elimination is a1,4-elimination illustrated as

In the elimination mechanism, the illustrated proton H is removed by abase; in aqueous media, the base is typically hydroxide ion such thatthe rate of elimination is determined by the pH of the medium. Underphysiological conditions, the pH of the fluid surrounding and permeatingthe hydrogel appears to be the predominant factor controlling the rateof elimination. Thus, when X and Y represent chains within a polymermatrix located in a physiological environment, pH-dependent eliminationresults in disruption of the bond between X and Y and subsequentbiodegradation of the polymer matrix in a process which does not requirethe action of enzymes.

By “a moiety capable of being cleaved by elimination under physiologicalconditions” is meant a structure comprising a group H—C—(CH═CH)_(m)—C—Xwherein m is 0 or 1 and X is a leaving group, wherein an eliminationreaction as described above to remove the elements of HX can occur at arate such that the half-life of the reaction is between 1 and 10,000hours under physiological conditions of pH and temperature. Preferably,the half-life of the reaction is between 1 and 5,000 hours, and morepreferably between 1 and 1,000 hours, under physiological conditions ofpH and temperature. By physiological conditions of pH and temperature ismeant a pH of between 7 and 8 and a temperature between 30 and 40° C.

It should be noted that when ranges are given in the presentapplication, such as 1-1,000 hours, the intermediate interval numbersshould be considered as disclosed as if specifically and explicitly setforth. This avoids the necessity of long list of numbers and applicantsclearly intend to include any arbitrary range between the outerboundaries. For example, the range 1-1,000 also includes 1-500 and 2-10.

By hydrogel is meant a three-dimensional, predominantly hydrophilicpolymeric network comprising a large quantity of water, formed bychemical or physical crosslinking of natural or synthetic homopolymers,copolymers, or oligomers. Hydrogels may be formed through crosslinkingpolyethylene glycols (considered to be synonymous with polyethyleneoxides), polypropylene glycols, poly(N-vinylpyrrolidone),polymethacrylates, polyphosphazenes, polylactides, polyacrylamides,polyglycolates, polyethylene imines, agarose, dextran, gelatin,collagen, polylysine, chitosans, alginates, hyaluronans, pectin,carrageenan. The polymer may be a multi-armed polymer as illustratedbelow.

Hydrogels may also be environment-sensitive, for example being liquidsat low temperature but gelling at 37° C., for example hydrogels formedfrom poly(N-isopropylacrylamide).

By mesoporous hydrogel is meant a hydrogel having pores betweenapproximately 1 nm and approximately 100 nm in diameter. The pores inmesoporous hydrogels are sufficiently large to allow for free diffusionof biological molecules such as proteins. By macroporous hydrogel ismeant a hydrogel having pores greater than approximately 100 nm indiameter. By microporous hydrogel is meant a hydrogel having pores lessthan approximately 1 nm in diameter.

By reactive polymer and reactive oligomer is meant a polymer or oligomercomprising functional groups that are reactive towards other functionalgroups, most preferably under mild conditions compatible with thestability requirements of peptides, proteins, and other biomolecules.Suitable functional groups found in reactive polymers includemaleimides, thiols or protected thiols, alcohols, acrylates,acrylamides, amines or protected amines, carboxylic acids or protectedcarboxylic acids, azides, alkynes including cycloalkynes, 1,3-dienesincluding cyclopentadienes and furans, alpha-halocarbonyls, andN-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, or nitrophenyl estersor carbonates.

By functional group capable of connecting to a reactive polymer is meanta functional group that reacts to a corresponding functional group of areactive polymer to form a covalent bond to the polymer. Suitablefunctional groups capable of connecting to a reactive polymer includemaleimides, thiols or protected thiols, acrylates, acrylamides, aminesor protected amines, carboxylic acids or protected carboxylic acids,azides, alkynes including cycloalkynes, 1,3-dienes includingcyclopentadienes and furans, alpha-halocarbonyls, andN-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, or nitrophenyl estersor carbonates.

By biodegradable hydrogel is meant a hydrogel that loses its structuralintegrity through the cleavage of component chemical bonds underphysiological conditions of pH and temperature. Biodegradation may beenzymatically catalyzed or may be solely dependent upon environmentalfactors such as pH and temperature. Biodegradation results in formationof fragments of the polymeric network that are sufficiently small to besoluble and thus undergo clearance from the system through the usualphysiological pathways.

By crosslinking reagent is meant a compound comprising at least twofunctional groups that are capable of forming covalent bonds with one ormore reactive polymers or oligomers. Typically, the reactive polymers oroligomers are soluble, and crosslinking results in formation of aninsoluble three-dimensional network or gel. The two functional groups ofthe crosslinking reagent may be identical (homobifunctional) ordifferent (heterobifunctional). The functional groups of theheterobifunctional crosslinking reagent are chosen so as to allow forreaction of one functional group with a cognate group of the reactivepolymer or oligomer and reaction of the second functional group with acognate group of the same or a different reactive polymer or oligomer.The two functional groups of a bifunctional crosslinking reagent arechosen so that they are not reactive with themselves, i.e., are notcognates.

Examples of cognate reactive pairs of functional groups include:

-   -   Azide+acetylene, cyclooctyne, maleimide    -   Thiol+maleimide, acrylate, acrylamide, vinylsulfone,        vinylsulfonamide, halocarbonyl    -   Amine+carboxylic acid, activated carboxylic acid    -   Maleimide+1,3-diene, cyclopentadiene, furan

Thus, as one example a heterobifunctional crosslinking reagent may beprepared having an azide and an amine group, but not an azide and acyclooctyne group.

“Substituted” means an alkyl, alkenyl, alkynyl, aryl, or heteroarylgroup comprising one or more substituent groups in place of one or morehydrogen atoms. Substituent groups may generally be selected fromhalogen including F, Cl, Br, and I; lower alkyl including linear,branched, and cyclic; lower haloalkyl including fluoroalkyl,chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy includinglinear, branched, and cyclic; SH; lower alkylthio including linear,branched, and cyclic; amino, alkylamino, dialkylamino, silyl includingalkylsilyl, alkoxysilyl, and arylsilyl; nitro; cyano; carbonyl;carboxylic acid, carboxylic ester, carboxylic amide; aminocarbonyl;aminoacyl; carbamate; urea; thiocarbamate; thiourea; ketone; sulfone;sulfonamide; aryl including phenyl, naphthyl, and anthracenyl;heteroaryl including 5-member heteroaryls including as pyrrole,imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole,thiadiazole, triazole, oxadiazole, and tetrazole, 6-member heteroarylsincluding pyridine, pyrimidine, pyrazine, and fused heteroarylsincluding benzofuran, benzothiophene, benzoxazole, benzimidazole,indole, benzothiazole, benzisoxazole, and benzisothiazole.

The properties of R¹ and R² may be modulated by the optional addition ofelectron-donating or electron-withdrawing substituents. By the term“electron-donating group” is meant a substituent resulting in a decreasein the acidity of the R¹R²CH; electron-donating groups are typicallyassociated with negative Hammett σ or Taft σ* constants and arewell-known in the art of physical organic chemistry. (Hammett constantsrefer to aryl/heteroaryl substituents, Taft constants refer tosubstituents on non-aromatic moieties.) Examples of suitableelectron-donating substituents include but are not limited to loweralkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino,and silyl. Similarly, by “electron-withdrawing group” is meant asubstituent resulting in an increase in the acidity of the R¹R²CH group;electron-withdrawing groups are typically associated with positiveHammett σ or Taft σ* constants and are well-known in the art of physicalorganic chemistry. Examples of suitable electron-withdrawingsubstituents include but are not limited to halogen, difluoromethyl,trifluoromethyl, nitro, cyano, C(═O)—R^(X), wherein R^(X) is H, loweralkyl, lower alkoxy, or amino, or S(O)_(m)R^(Y), wherein m=1-2 and R^(Y)is lower alkyl, aryl, or heteroaryl. As is well-known in the art, theelectronic influence of a substituent group may depend upon the positionof the substituent. For example, an alkoxy substituent on the ortho- orpara-position of an aryl ring is electron-donating, and is characterizedby a negative Hammett σ constant, while an alkoxy substituent on themeta-position of an aryl ring is electron-withdrawing and ischaracterized by a positive Hammett σ constant. A table of Hammett σ andTaft σ* constants values is given below.

Substituent σ(meta) σ(para) σ* H 0.00 0.00 0.49 CH₃ −0.07 −0.17 0 C₂H₅−0.07 −0.15 −0.10 n-C₃H₇ −0.07 −0.13 −0.115 i-C₃H₇ −0.07 −0.15 −0.19n-C₄H₉ −0.08 −0.16 −0.13 t-C₄H₉ −0.10 −0.20 −0.30 H₂C═CH 0.05 −0.02 0.56C₆H₅ 0.06 −0.01 0.60 CH₂Cl 0.11 0.12 1.05 CF₃ 0.43 0.54 2.61 CN 0.560.66 3.30 CHO 0.35 0.42 COCH₃ 0.38 0.50 1.65 CO₂H 0.37 0.45 2.08Si(CH₃)₃ −0.04 −0.07 −0.81 CH₂Si(CH₃)₄ −0.16 −0.22 −0.25 F 0.34 0.063.21 Cl 0.37 0.23 2.96 Br 0.39 0.23 2.84 I 0.35 0.18 2.46 OH 0.12 −0.371.34 OCH₃ 0.12 −0.27 1.81 OCH₂CH₃ 0.10 −0.24 1.68 OCF₃ 0.40 0.35 SH 0.250.15 1.68 SCH₃ 0.15 0.00 1.56 NO₂ 0.71 0.78 4.0 NO 0.62 0.91 NH₂ −0.16−0.66 0.62 NHCHO 0.19 0.00 NHCOCH₃ 0.07 −0.15 1.40 N(CH₃)₂ −0.15 −0.830.32 N(CH₃)⁺ 0.88 0.82 4.55 CCl₃ 0.47 2.65 CO₂CH₃ 0.32 0.39 2.00 CH₂NO₂1.40 CH₂CF₃ 0.92 CH₂OCH₃ 0.52 CH₂Ph 0.46 0.26 Ph 0.06 −0.01 0.60

“Alkyl”, “alkenyl”, and “alkynyl” include linear, branched or cyclichydrocarbon groups of 1-8 carbons or 1-6 carbons or 1-4 carbons whereinalkyl is a saturated hydrocarbon, alkenyl includes one or morecarbon-carbon double bonds and alkynyl includes one or morecarbon-carbon triple bonds. Unless otherwise specified these contain1-6C.

“Aryl” includes aromatic hydrocarbon groups of 6-18 carbons, preferably6-10 carbons, including groups such as phenyl, naphthyl, andanthracenyl. “Heteroaryl” includes aromatic rings comprising 3-15carbons containing at least one N, O or S atom, preferably 3-7 carbonscontaining at least one N, O or S atom, including groups such aspyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.

“Halogen” includes fluoro, chloro, bromo and iodo.

“Maleimido” is a group of the formula

The terms “protein” and “peptide” are used interchangeably regardless ofchain length, and these terms further include pseudopeptides whichcomprise linkages other than amide linkages, such as CH₂NH₂ linkages aswell as peptidomimetics.

The terms “nucleic acids” and “oligonucleotides” are also usedinterchangeably regardless of chain length. The nucleic acids oroligonucleotides may be single-chain or duplexed or may be DNA, RNA, ormodified forms thereof with altered linkages, such as phosphodiesters,phosphoramidates, and the like. For both the proteins and nucleic acidsuseful as drugs in the invention, these terms also include those withside chains not found in nature in the case of proteins and bases notfound in nature in the case of nucleic acids.

Small molecules in the context of drugs is a term well understood in theart, and is meant to include compounds other than proteins and nucleicacids that either are synthesized or are isolated from nature and ingeneral do not resemble proteins or nucleic acids. Typically, they havemolecular weights <1,000, although there is no specific cutoffrecognized. Nevertheless, the term is well understood in the fields ofpharmacology and medicine.

The present invention provides crosslinking reagents comprising a moietycapable of being cleaved by elimination under physiological conditionsand further comprising reactive groups capable of forming covalent bondswith reactive polymers. In one embodiment, the crosslinking reagents areof formula (1)

m is 0 or 1;

X comprises a functional group capable of connecting to a reactivepolymer that is amenable to elimination from the linker underphysiological conditions and a second reactive group Z² that couples toa reactive polymer;

wherein at least one of R¹, R², and R⁵ comprises a first functionalgroup Z¹ capable of connecting to a polymer;

at least one or both R¹ and R² is independently CN; NO2;

-   -   optionally substituted aryl;    -   optionally substituted heteroaryl;    -   optionally substituted alkenyl;    -   optionally substituted alkynyl;    -   COR³ or SOR³ or SO₂R³ wherein        -   R³ is H or optionally substituted alkyl;        -   aryl or arylalkyl, each optionally substituted;        -   heteroaryl or heteroarylalkyl, each optionally substituted;            or        -   OR⁹ or NR⁹ ₂ wherein each R⁹ is independently H or            optionally substituted alkyl, or both R⁹ groups taken            together with the nitrogen to which they are attached form a            heterocyclic ring;

SR⁴ wherein

-   -   R⁴ is optionally substituted alkyl;    -   aryl or arylalkyl, each optionally substituted; or    -   heteroaryl or heteroarylalkyl, each optionally substituted;

wherein R¹ and R² may be joined to form a 3-8 membered ring; and

wherein one and only one of R¹ and R² may be H or may be alkyl,arylalkyl or heteroarylalkyl, each optionally substituted; and

each R⁵ is independently H or is alkyl, alkenylalkyl, alkynylalkyl,(OCH₂CH₂)_(p)O-alkyl, wherein p=1-1000, aryl, arylalkyl, heteroaryl orheteroarylalkyl, each optionally substituted.

The crosslinking reagents of formula (1) comprise a moiety capable ofbeing cleaved by elimination under physiological conditions. Thus,hydrogels formed using crosslinking reagents of formula (1) arebiodegradable under physiological conditions. The elimination mechanismis dependent upon the pH and temperature of the medium. While thecrosslinking reagents are stable towards cleavage by elimination at lowpH and temperature, at physiological values of pH (approximately 7.4)and temperature (approximately 37° C.) the elimination occurs at a ratethat is controlled primarily by the R¹ and R² groups, and to a lesserdegree by the R⁵ groups.

The rates of the elimination reaction are predictable based on thestructures of the R¹, R², and R⁵ groups. Electron-withdrawing R¹ and R²groups accelerate the elimination reaction, while electron-donating R¹and R² groups retard the elimination reaction, such that the ratesobtained may be varied so as to provide linkers having half-lives forelimination from minutes to years. Alkyl R⁵ groups slow the eliminationreaction slightly relative to aryl R⁵ groups. By changing the R¹ and R²groups it is thus possible to control the rate at which the eliminationoccurs, and consequently the biodegradation rate of the hydrogel can becontrolled over a wide range. Hydrogels formed using crosslinkingreagents of formula (1) are thus expected to find use in applicationswhere a temporary gel matrix is required, for example as carriers ordepots for drug delivery or as temporary scaffolds for tissueregeneration.

Embodiments of X

X comprises a functional group capable of connecting to a reactivepolymer and is also amenable to elimination under physiologicalconditions. Typically, the resulting acid HX will have a pK_(a) of 10 orless, preferably a pK_(a) of 8 or less. Examples of suitable X groupsthus include carbonates, carbonyl halides, carbamates, thioethers,esters, and optionally substituted phenols. In one embodiment of theinvention, X is an activated carbonate such as succinimidyl carbonate,sulfosuccinimidyl carbonate, or nitrophenyl carbonate. In anotherembodiment of the invention, X is a carbonyl halide such as O(C═O)Cl orO(C═O)F. In another embodiment of the invention, X is a carbamate of theformula

wherein T* is O, S or NR⁶ wherein R⁶ is H, optionally substituted alkyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, or optionally substitutedheteroarylalkyl; z is 1-6; and Y is absent or is OR⁷ or SR⁷, wherein R⁷is optionally substituted alkylene, optionally substituted phenylene or(OCH₂CH₂)_(p), wherein p=1-1000, and Z² is a functional group capable ofconnecting with a reactive polymer. In one particular embodiment of theinvention, Y is (OCH₂CH₂)_(p), wherein p=1-1000; or Y is (OCH₂CH₂)_(p),wherein p=1-100; or Y is (OCH₂CH₂)_(p), wherein p=1-10.

In another embodiment, X is OR⁷ or SR⁷, wherein R⁷ is optionallysubstituted alkylene, optionally substituted phenylene or (OCH₂CH₂)_(p),wherein p=1-1000, and Z² is a functional group capable of connectingwith a reactive polymer.

In certain embodiments, the invention provides crosslinking reagents offormula (1) wherein R⁵ is the substituent among R¹, R² and R⁵ thatfurther comprises a functional group capable of connecting to a polymer.In more particular embodiments, the invention provides crosslinkingreagents of formula (1) wherein one of R⁵ further comprises a functionalgroup capable of connecting to a polymer and the other R⁵ is H.

Thus, the invention provides crosslinking reagents of formula (1a)

wherein m is 0-1; r is 2-8; and R¹, R², R⁵, m, X, and Z are as definedabove. In a more particular embodiment, the invention providescrosslinking reagents of formula (1a) wherein R⁵ is H. In an even moreparticular embodiment, the invention provides crosslinking reagents offormula (1a) wherein R¹ is CN or R⁸SO₂, wherein R⁸ is optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, or OR⁹ or NR⁹ ₂ wherein each R⁹ is independently H oroptionally substituted alkyl, or both R⁹ groups taken together with thenitrogen to which they are attached form a heterocyclic ring; R² and R⁵are H, and m=0.

In another embodiment, the invention provides crosslinking reagents offormula (1a) wherein X is of the formula

wherein T* is O, S or NR⁶ wherein R⁶ is H, optionally substituted alkyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, or optionally substitutedheteroarylalkyl; z is 1-6; and Y is absent or is OR⁷ or SR⁷, wherein R⁷is optionally substituted alkylene, optionally substituted phenylene or(OCH₂CH₂)_(p), wherein p=1-1000, and Z² is a functional group capable ofconnecting with a reactive polymer. In one particular embodiment of theinvention, Y is (OCH₂CH₂)_(p), wherein p=1-1000; or Y is (OCH₂CH₂)_(p),wherein p=1-100; or Y is (OCH₂CH₂)_(p), wherein p=1-10.

In another embodiment of the invention, X is OR⁷ or SR⁷, wherein R⁷ isoptionally substituted alkylene, optionally substituted phenylene or(OCH₂CH₂)_(p), wherein p=1-1000, and Z² is a functional group capable ofconnecting with a reactive polymer.

In one embodiment, the invention provides crosslinking reagents offormula (1b)

wherein m is 0-1 and R¹, R², R⁵, m, X, and Z² are as defined above. In amore particular embodiment, the invention provides crosslinking reagentsof formula (1b) wherein R⁵ is H. In an even more particular embodiment,the invention provides crosslinking reagents of formula (1b) wherein R¹is CN or R⁸SO₂, wherein R⁸ is optionally substituted alkyl, optionallysubstituted aryl, optionally substituted heteroaryl, or OR⁹ or NR⁹ ₂wherein each R⁹ is independently H or optionally substituted alkyl, orboth R⁹ groups taken together with the nitrogen to which they areattached form a heterocyclic ring; R² and R⁵ are H, and m=0.

Methods for preparation of compounds of formula (1) wherein X is OH, Cl,or O-succinimidyl has been previously disclosed in patent publicationsWO2009/158668, WO2011/140393 and WO2011/140392. Compounds of formula (1)wherein X is a carbamate of the formula

may be prepared from compounds of formula (1) wherein X is Cl orO-succinimidyl by reaction with amines of the formula.R⁶—NH—(CH₂)_(z)Y—Z² using methods illustrated in the working examplesbelow.

In another embodiment of the invention, multivalent crosslinkingreagents of formula (2) are provided

wherein at least one of R¹, R² and R⁵ comprises a functional group Z¹capable of connecting to a polymer, and are otherwise defined as informula (1);

wherein

m is 0 or 1;

n is 1-1000;

s is 0-2;

t is 2, 4, 8, 16 or 32,

W is O(C═O)O, O(C═O)NH, O(C═O), S,

and

Q is a core group having a valency=t, wherein t=2, 4, 8, 16, or 32.

The core group Q is a group of valency=t which connects the multiplearms of the crosslinking reagent. Typical examples of Q include C(CH₂)₄(t=4), wherein the multi-arm reagent is prepared based on apentaerythritol core; (t=8), wherein the multi-arm reagent is preparedbased on a hexaglycerin core; and (t=8), wherein the multi-arm reagentis prepared based on a tripentaerythritol core.

Compounds of formula (2) may be prepared by the reaction of a multi-armpolyethylene glycol with a reagent of formula (1). A variety ofmulti-arm polyethyleneglycols are commercially available, for examplefrom NOF Corporation and JenKem Technologies.

In one particular embodiment of the invention, t is 4. In anotherembodiment of the invention, t is 8.

Preparation of Hydrogels

In another aspect the invention provides methods for preparingbiodegradable hydrogels comprising either simultaneously or sequentiallycontacting at least one reactive polymer and a cleavable crosslinkercompound wherein said cleavable crosslinker compound comprises afunctional group that reacts with the reactive polymer and a moiety thatcleaves by elimination under physiological conditions.

In one embodiment of the invention, biodegradable hydrogels are formedby reaction of a single reactive polymer and a cleavable crosslinkercompound wherein said cleavable crosslinker compound comprises afunctional group that reacts with the reactive polymer and a moiety alsoincluding a functional group that reacts with a reactive polymer thatcleaves by elimination under physiological conditions. In thisembodiment, the reactive polymer will be multi-valent, so as to allowformation of nodes in the three-dimensional hydrogel matrix. As oneillustration of this method, a multi-arm PEG wherein each arm isterminated with a reactive functional group Z³ as defined below isallowed to react with a crosslinker reagent of formula (1) or (2) so asto form a hydrogel. Multi-arm PEGs are commercially available in avariety of sizes and with a variety of reactive functional groups, forexample from NOF Corporation and JenKem Technologies. As anotherillustration of this method, a linear polymer which comprises multiplecopies of a reactive functional group Z³ is allowed to react with acrosslinker reagent of formula (1) or (2) so as to form a hydrogel.Illustrations of such linear polymers comprising multiple Z³ groups arehyaluronic acid, carboxymethyl cellulose, polyvinyl alcohol,poly(2-hydroxyethyl methyacrylate), dextran, collagen, chitosan,alginate, and agarose.

In another embodiment the invention provides methods for the formationof biodegradable hydrogels through reaction of a first reactive polymer,a second reactive polymer, and a cleavable crosslinker compound thatcomprises a first functional group that reacts with the first reactivepolymer, a second functional group that reacts with the second polymer,and a moiety that cleaves by elimination under physiological conditions.The first and second functional groups may be the same or different. Forthe formation of a three-dimensional gel network the reactive components(first reactive polymer, second reactive polymer if any) will bemulti-armed and thus serve to form nodes in the gel matrix. In preferredembodiments of the invention, this node-forming reactive componentcomprises at least 3 arms and more preferably at least 4 arms.

In each embodiment the reactive polymers may be homopolymeric orcopolymeric polyethylene glycols, polypropylene glycols,poly(N-vinylpyrrolidone), polymethacrylates, polyphosphazenes,polylactides, polyacrylamides, polyglycolates, polyethylene imines,agaroses, dextrans, gelatins, collagens, polylysines, chitosans,alginates, hyaluronans, pectins, or carrageenans that either comprisesuitable reactive functionalities in their native state or have beenderivatized so as to comprise suitable reactive functionalities. Typicalsuitable reactive functionalities include maleimides, thiols orprotected thiols, alcohols, acrylates, acrylamides, amines or protectedamines, carboxylic acids or protected carboxylic acids, azides, alkynesincluding cycloalkynes, 1,3-dienes including cyclopentadienes andfurans, alpha-halocarbonyls, and N-hydroxysuccinimide orN-hydroxysulfosuccinimide esters or carbonates. Native polymers that donot comprise an effective multiplicity of reactive groups can betransformed by reaction with reagents that introduce an effectivemultiplicity of reactive groups prior to formation of the hydrogel.

In some embodiments, polymers include multivalent branched structures ofthe formula [Z³—(CH₂)_(s)—(CH₂CH₂O)_(n)]_(t)Q, wherein Z³ is a reactivefunctional group selected from the options set forth above for Z¹ andZ², s is 0-2, Q is a multivalent core group having valency t, wherein tis 2, 4, 8, 16 or 32. The value of n can be 10-1000 or intermediatevalues such as 20, 50, 100, etc. This listing is intended to include allintermediate integers between 10 and 1000.

The gel forming reactions may be performed in a variety of suitablesolvents, for example water, alcohols, acetonitrile, or tetrahydrofuran,and are preferably performed in aqueous medium.

Formation of the hydrogels may be performed in a stepwise or a concertedfashion. Thus, in one embodiment of the invention, a first reactivepolymer is allowed to react with a crosslinking reagent of formula (1)or (2) so as to form an intermediate non-crosslinked combination, whichis optionally isolated. This non-crosslinked combination is then allowedto react with the second reactive polymer to form the final crosslinkedgel. In another embodiment of the invention, the first reactive polymer,second reactive polymer, and crosslinking reagent of formula (1) or (2)are combined and allowed to react and form the hydrogel in a singleoperation.

In one embodiment, the invention provides methods for formation ofhydrogels by crosslinking a polymer with a crosslinking reagent offormula (1). Depending upon the functionality present, the polymer maybe in its native state or may be first derivatized using methods knownin the art to introduce functionality that is cross-reactive with thefunctionality on the compound of formula (1). In this embodiment, thetwo functional groups capable of reacting with a polymer on the compoundof formula (1) are typically the same. An example of this embodiment isillustrated in FIG. 1. As shown, a cleavable crosslinker of Formula (1)with two azide functional groups crosslinks a 4-armed polymer withcyclooctyne functional groups. Alternative gels with other embodimentsas noted above for Z¹, Z² and Z³ are prepared to provide similar oridentical results.

In another embodiment, the invention provides methods for formation ofhydrogels by crosslinking two differently substituted polymers one ofwhich comprises a crosslinker susceptible to elimination. Two examplesof this embodiment are illustrated in FIGS. 2A and 2B. FIG. 2A showscrosslinking a first 4-arm polymer wherein each arm is terminated with acyclooctyne (CO) with a second 4-arm polymer wherein each arm isterminated with a beta-eliminative linker azide compound of formula (1)(L₂-N₃) which is thus a 4-arm compound of formula (2). The resulting 4×4hydrogel comprises a beta-eliminative linker in each crosslink. The gelthus contains alternating nodes derived from the 4-arm polymer and fromFormula (2).

As illustrated in

FIG. 2B, this method may also use polymers with a greater number ofarms. As shown, some of the arms of the 8-armed polymer may bederivatized to a drug through coupling to a compound of formula (3)shown below. In addition, or instead, one or more of the arms may becoupled to a marker compound, such as a fluorescent dye in order toevaluate the rate of disintegration of the gel as a function of theenvironmental conditions and/or as a function of the nature of R¹, R²and/or R⁵. This “erosion probe” permits design of gels with desireddisintegration rates.

In one aspect of such design, a drug may be simply included in the poresof the gel by forming the gel in the presence of the drug and thedelivery rate of the drug's controlled by appropriate choice ofsubstituents in the crosslinking compounds that result in gel formation.

Gels may also be prepared which contain drug both included in the poresand coupled to the polymer through a linkage as shown in formula (3)below. The rates of release from the linkage and from the pores can thenbe compared.

In the third alternative, the drug may be supplied simply in the form offormula (3) so that the release rate from the gel is determined solelyby the elimination reaction of the drug from the gel.

In another aspect, the invention provides hydrogels that are formedaccording to the above methods. These hydrogels may comprise a varietyof hydrophilic polymers, included as described above native or modifiedforms of polyethylene glycols, polypropylene glycols,poly(N-vinylpyrrolidone), polymethacrylates, polyphosphazenes,polylactides, polyacrylamides, polyglycolates, polyethylene imines,agaroses, dextrans, gelatins, collagens, polylysines, chitosans,alginates, hyaluronans, pectins, carrageenans, or the multi-armedpolymers illustrated, and are characterized by their crosslinking whichincludes at least one moiety capable of being cleaved by eliminationunder physiological conditions. These hydrogels are thus biodegradablethrough a pH-dependent process.

Through appropriate choice of reactants and stoichiometries, the poresize of the resulting hydrogels may be determined. The hydrogels of theinvention may be microporous, mesoporous, or macroporous, and may have arange of biodegradation rates that are determined by the nature of thecrosslinking reagents used in their preparation.

The hydrogels of the invention may also comprise residual reactivegroups that were not consumed in the gelling process, either through thestoichiometry chosen, through incomplete crosslinking, or throughincorporation of functional groups that do not participate in thegelling process due to orthogonal reactivity. These residual reactivegroups may be used to further modify the resulting hydrogel, for exampleby covalent attachment of drugs or prodrugs. In one embodiment of theinvention, the residual reactive groups are used to attach prodrugscomprising a drug attached to a linker that subsequently releases thedrug from the hydrogel matrix. In a more particular embodiment of theinvention, release of the drug from the hydrogel matrix occurs via anelimination mechanism. The use of eliminative linkers for drugconjugation is described, for example, in PCT publications WO2009/158668and WO2011/140393, which are hereby incorporated by reference.

One embodiment of drug-releasing degradable hydrogels of the inventionis illustrated in FIG. 2B and exemplified in working Examples 29 and 33below. Reaction of a subset of the functional groups on a first polymerwith a releasable linker-drug, wherein the linker comprises a firstmodulator group that controls the rate of drug release, provides anintermediate drug-loaded polymer; the residual functional groups arereacted with a crosslinking reagent of formula (1) or (2) comprising asecond modulator group that controls the rate of hydrogel degradation toprovide a drug-loaded degradable hydrogel. By appropriate selection ofthe modulator groups present on the drug linker and on the crosslinkingreagent, the rates of drug release and of hydrogel degradation can becontrolled. In one method of the invention, the first polymer is treatedwith the linker-drug in a first step; the intermediate drug-loadedpolymer is optionally isolated; and the hydrogel is formed by reactionwith the crosslinker reagent in a separate step. In a second method ofthe invention, the first polymer, linker-drug, and crosslinker reagentare combined in a single step. If all reactive functionalities on thepolymers are not consumed by either connection to linker-drug orcrosslinking, the excess functionalities may optionally be capped byreaction with suitable reagents. For example, excess cyclooctynes may becapped by reaction with short PEG-azides such as azido-heptaethyleneglycol.

Thus, in one embodiment of the invention, a method for forming adrug-releasing degradable hydrogels is provided consisting of the stepsof:

(a) reacting a first multivalent polymer comprising reactivefunctionalities with a substoichiometric amount of a linker-drug havingthe formula (3)

wherein m, R¹, R², and R⁵ may have the embodiments listed for these inFormulas (1) and (2) although, of course, independently selected, sothat a gel that contains both residues of formula (1) or (2) and Formula(3) need not comprise the same substituents of these notations, D is theresidue of a drug and Y, in this case, is NH or NBCH₂, wherein B is H,alkyl, arylalkyl, heteroaryl, or heteroarylalkyl, each optionallysubstituted, wherein at least one of R¹, R², R⁵ is substituted with afunctional group corresponding to Z¹ reactive with a functional group onthe first polymer;

so as to form a drug-loaded first polymer;

(b) optionally isolating the drug-loaded first polymer; and

(c) crosslinking the remaining reactive functionalities on thedrug-loaded first polymer with a compound of formula (1) or formula (2)so as to form a hydrogel.

The preparation of linker-drugs of formula (3) is detailed in PCTpublications WO2009/158668 and WO/2011/140393, which are herebyincorporated by reference.

The linked drug D may be a small molecule or a polypeptide, includingpeptides and proteins. Working Example 32 below details the preparationof a drug-releasing degradable hydrogel wherein D is the peptideexenatide, which has the sequence:H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂ (SEQ ID NO:1).

In one embodiment of the invention, the exenatide peptide is coupled tothe linker via an amino group to provide

wherein R¹, R², R⁵, and m are as defined for formula (3) above. Incertain embodiments, m=0, R² is H, one R⁵ is H, and the other R⁵ is(CH₂)_(n)Y wherein n=1-6 or CH₂(OCH₂CH₂)_(p)Y wherein p=1-1000 and Y isa group comprising an N₃, SH, S^(t)Bu, maleimide, 1,3-diene,cyclopentadiene, furan, alkyne, cyclooctyne, acrylate, acrylamide, vinylsulfone, or vinyl sulfonamide group. In certain embodiments of theinvention, R¹ is CN or SO₂R³, wherein R³ is optionally substitutedalkyl, optionally substituted aryl, optionally substituted heteroaryl,OR⁹, or N(R⁹)₂, wherein each R⁹ is independently H, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, and wherein N(R⁹)₂ may form a heterocyclic ring. The linkermay be coupled to any free amino group on the peptide, i.e., theN-terminal amine or any side-chain amine such as the epsilon-aminogroups of lysine.

In one specific embodiment of the invention, the linker-drug of formula(3) comprises a reactive azide group on one R⁵. A substoichiometricamount of the linker-drug is thus reacted with a multi-arm polymercomprising reactive cyclooctyne groups at the terminus of each arm.Examples of reactive cyclooctyne groups include those effective incopper-free 1,3-dipolar cycloaddition reactions with azides, includingfor example dibenzocyclooctynes, dibenzoazacyclooctynes (DBCO),difluorocyclooctynes (DIFO), and strained bicyclic cyclooctynes such asbicyclononynes (BCN).

In one embodiment of the invention, the first polymer comprises at least8 arms, each arm terminated with a reactive functional group. As shownin FIG. 2B, 3 arms of the first polymer are used for crosslinking tocompounds of formula (1) or (2). In a preferred embodiment of theinvention, at least 4 arms of the first polymer are used forcrosslinking to compounds of formula (1) or (2). Thus, thesubstoichiometric amount of linker-drug used may range from 0.01 to 5molar equivalents relative to the first polymer, leading to loading of0.01 to 5 molecules of drug D per 8-arm first polymer. In one embodimentof the invention, the substoichiometric amount of linker-drug used mayrange from 0.1 to 5 molar equivalents relative to the first polymer. Inanother embodiment of the invention, the substoichiometric amount oflinker-drug used may range from 1 to 5 molar equivalents relative to thefirst polymer.

Thus, in certain embodiments of the invention, an exenatide-releasingdegradable hydrogel is prepared by reacting a multivalent first polymercomprising a cyclooctyne group at the terminus of each arm with asubstoichiometric amount of a linker-drug of formula (4)

-   -   wherein R¹═CN; NO₂;    -   optionally substituted aryl;    -   optionally substituted heteroaryl;    -   optionally substituted alkenyl;    -   optionally substituted alkynyl;    -   COR³ or SOR³ or SO₂R³ wherein    -   R³ is H or optionally substituted alkyl;    -   aryl or arylalkyl, each optionally substituted;    -   heteroaryl or heteroarylalkyl, each optionally substituted; or    -   OR⁹ or NR⁹ ₂ wherein each R⁹ is independently H or optionally        substituted alkyl, or both R⁹ groups taken together with the        nitrogen to which they are attached form a heterocyclic ring; or    -   SR⁴ wherein    -   R⁴ is optionally substituted alkyl;    -   aryl or arylalkyl, each optionally substituted; or    -   heteroaryl or heteroarylalkyl, each optionally substituted;    -   so as to form an exenatide-loaded first polymer, which is        optionally isolated, for example by precipitation,        size-exclusion or ion-exchange chromatography, or other methods        known in the art. In specific embodiments of the invention, R¹        in formula (4) is CN or SO₂R³.

The exenatide-loaded first polymer is then reacted with a cleavablecompound of formula (1) or (2) to form the exenatide-releasingdegradable hydrogel. In certain embodiments of the invention, theexenatide-releasing first polymer is an 8-arm polyethylene glycol, andthe cleavable compound used for hydrogel formation is a compound offormula (2). In certain embodiments of the invention, the cleavablecompound used for hydrogel formation is a compound of formula (2)wherein m is 0, n is 10-150, s is 0, t is 4, and Q is C(CH₂)₄.

As described above, the rates of drug release and of hydrogeldegradation are controlled primarily by choice of the R¹ and R² groupson the drug-linkers and crosslinkers, respectively. The chosen rate ofdrug release is typically determined by the desired pharmacokinetics ofthe drug, e.g. the maximal and/or minimal concentrations of free drugover the duration of administration, as has been described in Santi etal PNAS (2012) (submitted) and in co-pending PCT application numberPCT/US2012/054293 (filed 7 Sep. 2012), both of which are herebyincorporated by reference. The R¹ and R² groups on the compounds offormula (I) and (II) are then chosen to provide the optimal rate ofhydrogel degradation in order to supply the needed amount of free drugover the duration of administration while minimizing the lifetime of thedegradable hydrogel in the body.

In another embodiment of the invention, drug-releasing degradablehydrogels are prepared by a method wherein a multi-arm first polymerwherein each arm is terminated by a group comprising at least twoorthogonal functional groups is reacted with a linker-drug of formula(3) wherein the linker-drug comprises a functional group that reactswith only one of the orthogonal functional groups present on the firstpolymer. The remaining orthogonal functional group on the resultingdrug-loaded first polymer is used to form a hydrogel by reaction with acompound of formula (1) or (2) wherein these compounds comprise afunctional group that reacts with only one the remaining orthogonalfunctional groups present on the drug-loaded first polymer. This methodis advantageous in that it should provide drug-releasing degradablehydrogels of more regular structure than those formed by stoichiometriccontrol of components. This method is illustrated in working Example 37below. The multi-arm first polymer wherein each arm is terminated by agroup comprising at least two orthogonal functional groups can beprepared from multi-arm polymers wherein each arm terminates with asingle functional group by condensation with an appropriatemulti-functional adapter. This is illustrated in FIG. 11.

The hydrogels of the invention may be prepared in vitro, then implantedas required. The gels may be cast into specific shapes, or may beprepared as microparticulate or microspherical suspensions forinjection. Alternatively, the hydrogels may be formed by in situgelation, in which case pharmaceutically acceptable formulations of thehydrogel components are prepared; mixing of the components is followedby injection or application prior to gelation. Injection may besubcutaneous, intramuscular, intraocular, intratumoral, or intravenous.The hydrogels of the invention may be applied topically, for example byin situ gelation of the mixed components after application to the skinor to surgical wounds. The hydrogels of the invention may also beapplied as coatings on medical devices or surgical dressings.

All references cited herein are hereby incorporated by reference intheir entirety. The invention is further illustrated but not limited bythe following examples.

Example 1 Preparation of 6-Azidohexanal

(1) 6-Azido-1-hexanol: a mixture of 6-chloro-1-hexanol (25 g, 183 mmol)and sodium azide (32.5 g, 500 mmol) in 200 mL of water was heated atreflux for 20 h, then cooled to ambient temperature and extracted 3×with ethyl acetate. The combined extracts were washed with brine, driedover MgSO₄, filtered, and concentrated to yield the product as a paleyellow oil (28.3 g).

(2) 6-Azidohexanal: Solid trichloroisocyanuric acid (4.3 g) was added insmall portions to a vigorously stirred mixture of 6-azido-1-hexanol(7.15 g), TEMPO (50 mg), and sodium bicarbonate (5.0 g) indichloromethane (100 mL) and water (10 mL). The mixture was stirred foran additional 30 minutes after addition, then filtered through a pad ofCelite. The organic phase was separated and washed successively withsat. aq. NaHCO₃ and brine, then dried over MgSO₄, filtered, andconcentrated to provide the product (5.8 g), which was used withoutfurther purification.

Example 2 Preparation of ω-Azido-PEG-Aldehydes

Solid trichloroisocyanuric acid (60 mg) was added to a vigorouslystirred mixture of O-(2-azidoethyl) heptaethylene glycol (n=7; 250 mg),1 mg of TEMPO, 100 mg of NaHCO₃, 2 mL of CH₂Cl₂, and 0.2 mL of water.The mixture turned orange, and after approximately 30 minutes a whitesuspension was formed. TLC analysis (1:1 acetone/hexane) indicatedformation of a product that stained with phosphomolybdic acid. Themixture was diluted with 10 mL of CH₂Cl₂, dried by stirring with MgSO₄,filtered, and evaporated to yield the crude product. This was dissolvedin CH₂Cl₂ and loaded onto a 4-gm column of silica gel equilibrated inhexane, which was eluted sequentially with 25 mL each of hexane, 75:25hexane/acetone, 50:50 hexane/acetone, and 25:75 hexane/acetone.Product-containing fractions were combined and evaporated to provide thepurified product.

Example 3 Preparation of Azidoalcohols

A 1.6 M solution of n-butyllithium (3.1 mL, 5.0 mmol) in hexane wasadded dropwise to a stirred solution of R—SO₂CH₃ (5.0 mmol) in anhydroustetrahydrofuran (THF) (15 mL) cooled to −78° C. After addition, thecooling bath was removed and the mixture was allowed to warm slowly to0° C. over approximately 30 min. The mixture was then cooled back to−78° C., and 6-azidohexanal (5.5 mmol) was added. After stirring for 15minutes, the cooling bath was removed and the mixture was allowed towarm. At the point where the mixture became clear, 5 mL of saturated aq.NH₄Cl was added and the mixture was allowed to continue warming toambient temperature. The mixture was diluted with ethyl acetate andwashed successively with water and brine, and then dried over MgSO₄,filtered, and evaporated to provide the crude product as an oil.Chromatography on silica gel using a gradient of ethyl acetate in hexaneprovided the purified products.

Compounds prepared according to this method include:

1-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptanol: from4-(trifluoromethyl)phenyl methyl sulfone (1.73 g, 94%): ¹H-NMR (400 MHz,CDCl₃): δ 8.08 (2H, d, J=8.4-Hz), 7.87 (2H, d, J=8.4-Hz), 4.21 (1H, m),3.25 (2H, t, J=6.8-Hz), 3.28 (1H, dd, J=8.8, 14.4-Hz), 3.20 (1H, dd,J=2.0, 14.4-Hz), 3.12 (1H, d, J=2.8-Hz), 1.58 (2H, m), 1.5-1.3 (6H, m);

1-(4-chlorophenylsulfonyl)-7-azido-2-heptanol; from 4-chlorophenylmethyl sulfone; colorless oil (1.49 g, 90% yield): ¹H-NMR (400 MHz,d₆-DMSO): δ 7.90 (2H, d, J=8.8-Hz), 7.70 (2H, d, J=8.8-Hz), 4.83 (1H, d,J=6-Hz), 3.86 (1H, m), 3.39 (2H, m), 3.29 (2H, t, J=6.8-Hz), 1.2-1.5(8H, m);

1-(phenylsulfonyl)-7-azido-2-heptanol; from phenyl methyl sulfone; paleyellow oil (1.25 g, 85%): ¹H-NMR (400 MHz, d₆-DMSO): δ 7.89 (2H, m),7.72 (1H, m), 7.63 (2H, m), 4.84 (1H, d J=6-Hz), 3.86 (1H, m), 3.33 (2H,m), 3.28 (2H, t, J=6.8-Hz), 1.47 (2H, m), 1.2-1.4 (6H, m);

1-(4-methylphenylsulfonyl)-7-azido-2-heptanol; from4-(methylsulfonyl)toluene; colorless oil (1.39 g, 85% yield): ¹H-NMR(400 MHz, d₆-DMSO): δ 7.76 (2H, d, J=6.4-Hz), 7.43 (2H, d, J=6.4-Hz),4.82 (1H, d, J=6-Hz), 3.85 (1H, m), 3.31 (2H, m), 3.28 (2H, t,J=6.8-Hz), 2.41 (3H, s), 1.4-1.5 (2H, m), 1.2-1.4 (6H, m);

1-(4-methoxyphenylsulfonyl)-7-azido-2-heptanol; from 4-methoxyphenylmethyl sulfone (1.53 g, 94% yield): ¹H-NMR (400 MHz, CDCl₃): δ 7.85 (2H,d, J=8.8-Hz), 7.04 (2H, d, J=8.8-Hz), 4.13 (1H, m), 3.90 (3H, s), 3.24(2H, t, J=6.8-Hz), 3.20 (1H, dd, J=8.8, 14.4-Hz), 3.14 (1H, dd, J=2.4,14.4-Hz), 2.47 (3H, s), 1.57 (2H, m), 1.5-1.3 (6H, m);

1-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptanol; from(2,4,6-trimethyl)phenyl methyl sulfone (1.30 g from 4.0 mmol reaction;96%): ¹H-NMR (400 MHz, CDCl₃): δ 6.99 (2H, s), 4.30 (1H, m), 3.49 (1H,d, J=2-Hz), 3.25 (2H, t, J=6.8-Hz), 3.18 (1H, d, J=1-Hz), 3.17 (1H, s),2.66 (6H, s), 2.31 (3H, s), 1.59 (2H, m), 1.5-1.3 (6H, m);

1-(morpholinosulfonyl)-7-azido-2-heptanol; from 1-morpholinomethylsulfonamide (1.36 g from 10 mmol reaction, 89%): ¹H-NMR (400 MHz,d₆-DMSO): δ 4.99 (1H, d, J=6.4 Hz), 3.88 (1H, m), 3.62 (4H, t,J=4.8-Hz), 3.32 (2H, t, J=6.8-Hz), 3.20-3.15 (6H, overlap), 1.53 (2H,m), 1.46-1.25 (6H, m); and

1-(methylsulfonyl)-7-azido-2-heptanol; from dimethylsulfone; colorlessoil (880 mg, 75%): ¹H-NMR (400 MHz, d₆-DMSO).

Example 4 Preparation of Azidoalcohols

A 1.6 M solution of n-butyllithium (3.1 mL, 5.0 mmol) in hexane is addeddropwise to a stirred solution of R—SO₂CH₃ (5.0 mmol) in anhydroustetrahydrofuran (THF) (15 mL) cooled to −78° C. After addition, thecooling bath is removed and the mixture is allowed to warm slowly to 0°C. over approximately 30 min. The mixture is then cooled back to −78°C., and ω-azido-heptaethylene glycol aldehyde (n=7, 1.2 g) is added.After stirring for 15 minutes, the cooling bath is removed and themixture is allowed to warm. At the point where the mixture becomesclear, 5 mL of sat. aq. NH₄Cl is added and the mixture is allowed tocontinue warming to ambient temperature. The mixture is diluted withethyl acetate and washed successively with water and brine, and thendried over MgSO₄, filtered, and evaporated to provide the crude product.Chromatography on silica gel provides the purified products.

Example 5 Preparation of Azido-Linker Chloroformates

Pyridine (160 μL) was added dropwise to a stirred solution of theazidoalcohol of Example 3 (1.0 mmol) and triphosgene (500 mg) in 15 mLof anhydrous THF. The resulting suspension was stirred for 10 minutes,then filtered and concentrated to provide the crude chloroformate as anoil.

Compounds prepared according to this method include:

-   1-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptyl chloroformate-   1-(4-chlorophenylsulfonyl)-7-azido-2-heptyl chloroformate;-   1-(phenylsulfonyl)-7-azido-2-heptyl chloroformate;-   1-(4-methylphenylsulfonyl)-7-azido-2-heptyl chloroformate;-   1-(4-methoxyphenylsulfonyl)-7-azido-2-heptyl chloroformate;-   1-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptyl chloroformate;-   1-(4-morpholinosulfonyl)-7-azido-2-heptyl chloroformate; and-   1-(methanesulfonyl)-7-azido-2-heptyl chloroformate.

Other chloroformates may be prepared according to this general method.

Example 6 Preparation of Azido-Linker Chloroformates

Pyridine (160 μL) is added dropwise to a stirred solution of theazidoalcohol of Example 4 (1.0 mmol) and triphosgene (500 mg) in 15 mLof anhydrous THF. The resulting suspension is stirred for 10 minutes,then filtered and concentrated to provide the crude chloroformate.

Example 7 Preparation of Azido-Linker Succinimidyl Carbonates

Pyridine (300 μL) was added dropwise to a stirred solution of thechloroformate of Example 5 (1.0 mmol) and N-hydroxysuccinimide (350 mg)in 15 mL of anhydrous THF. The resulting suspension was stirred for 10minutes, then filtered and concentrated to provide the crudesuccinimidyl carbonate. Purification by silica gel chromatographyprovided the purified product as an oil which spontaneouslycrystallized. Recrystallization could be effected using ethylacetate/hexane.

Compounds prepared according to this method include:

O-[1-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidylcarbonate: crystals from 40:60 ethyl acetate/hexane (280 mg, 55%):¹H-NMR (400 MHz, d₆-DMSO): δ 8.12 (2H, m), 8.04 (2H, m), 5.18 (1H, m),4.15 (1H, dd, J=9.2, 15.2), 3.96 (1H, dd, J=2.4, 15.2), 3.29 (2H, t,J=6.8), 2.80 (4H, s), 1.68 (2H, m), 1.47 (2H, m), 1.27 (4H, m);

O-[1-(4-chlorophenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidylcarbonate: crystals from 40:60 ethyl acetate/hexane (392 mg, 83%):¹H-NMR (400 MHz, d₆-DMSO): δ 7.85 (2H, m), 7.72 (2H, m), 5.14 (1H, m),4.04 (1H, dd, J=9.6, 15.6), 3.87 (1H, dd, J=2.4, 15.6), 3.29 (2H, t,J=6.8), 2.81 (4H, s), 1.68 (2H, m), 1.47 (2H, m), 1.27 (4H, m);

O-[1-(phenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidyl carbonate:crystals from 40:60 ethyl acetate/hexanes (391 mg, 89%): ¹H-NMR (400MHz, d₆-DMSO): δ 7.91 (2H, m), 7.76 (1H, m), 7.66 (2H, m), 5.12 (1H, m),3.96 (1H, dd, J=8.8, 15.2), 3.83 (1H, dd, J=2.8, 15.2), 3.29 (2H, t,J=6.8), 2.81 (4H, s), 1.69 (2H, m), 1.47 (2H, m), 1.27 (4H, m);

O-[1-(4-methylphenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidylcarbonate: crystals upon standing after chromatography (402 mg, 89%):¹H-NMR (400 MHz, d₆-DMSO): δ 7.77 (2H, d, J=8.0); 7.45 (2H, d, J=8.0);5.11 (1H, m), 3.90 (1H, dd, J=8.8, 15.2), 3.79 (1H, dd, J=1.8, 15.2),3.28 (2H, t, J=6.8), 2.81 (4H, s), 2.41 (3H, s), 1.68 (2H, m), 1.47 (2H,m), 1.27 (4H, m);

O-[1-(4-methoxyphenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidylcarbonate: crystals from 60:40 ethyl acetate/hexane (320 mg, 68%):¹H-NMR (400 MHz, d₆-DMSO): δ 7.81 (2H, d, J=8.8); 7.15 (2H, d, J=8.8);5.11 (1H, m), 3.87 (1H, dd, J=8.8, 15.2), 3.86 (3H, s), 3.76 (1H, dd,J=2.8, 15.2), 3.29 (2H, t, J=6.8), 2.80 (4H, s), 1.68 (2H, m), 1.47 (2H,m), 1.27 (4H, m);

O-[1-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidylcarbonate: colorless oil (458 mg, 95%): ¹H-NMR (400 MHz, d₆-DMSO): δ7.09 (2H, s), 5.20 (1H, m), 3.82 (1H, dd, J=8.4, 15.2-Hz), 3.67 (1H, dd,J=3.2, 15.2-Hz), 3.30 (2H, t, J=6.8-Hz), 2.79 (4H, s), 2.58 (6H, s),2.28 (3H, s), 1.75 (2H, m), 1.49 (2H, m), 1.30 (4H, m);

O-[1-(morpholinosulfonyl)-7-azido-2-heptyl]-O′-succinimidyl carbonate:crystals upon standing after chromatography (430 mg, 95%): (400 MHz,CDCl₃): δ 5.23 (1H, m), 3.77 (4H, dd, J=4.0, 5.6-Hz), 3.39 (1H, dd,J=6.4, 14.4-Hz), 3.31 (6H, overlap), 3.17 (1H, dd, J=4.8, 14.4-Hz), 2.85(4H, s), 1.88 (2H, m), 1.61 (2H, m), 1.45 (4H, m); and

O-[1-methylsulfonyl-7-azido-2-heptyl]-O′-succinimidyl carbonate:crystals upon standing after chromatography (360 mg, 95%): (400 MHz,CDCl₃): δ 5.32 (1H, m), 3.50 (1H, dd, J=7.2, 14.8-Hz), 3.29 (2H, t,J=6.8-Hz), 3.21 (1H, dd, J=0.8, 4.0, 14.8-Hz), 3.02 (3H, s), 2.85 (4H,s), 1.90 (2H, m), 1.62 (2H, m), 1.46 (4H, m).

Other succinimidyl carbonates may be prepared according to this generalmethod.

Example 8 Preparation of Azido-Linker Succinimidyl Carbonates

Pyridine (300 μL) is added dropwise to a stirred solution of thechloroformate of Example 6 (1.0 mmol) and N-hydroxysuccinimide (350 mg)in 15 mL of anhydrous THF. The resulting suspension is stirred for 10minutes, then filtered and concentrated to provide the crudesuccinimidyl carbonate. Purification by silica gel chromatographyprovides the purified product.

Example 9 Preparation of Azido-Linker Sulfosuccinimidyl Carbonates

A stirred suspension of sodium N-hydroxysuccinimide sulfonate (1 mmol)in N,N-dimethylformamide (10 mL) is treated with pyridine (3 mmol) and achloroformate of Example 7. After the suspension clears, the mixture isdiluted with ethyl acetate.

Example 10 Preparation of Amino-Linker Alcohols

A stirred solution of an azido-linker alcohol of Example 3 (R=phenyl; 1mmol) in 1 mL of tetrahydrofuran (THF) was treated with a 1.0 M solutionof trimethyl-phosphine in THF (1.2 mL) for 1 hour at ambienttemperature. Water (0.1 mL) was added, and the mixture was allowed tostir for an additional 1 hour, then the mixture was evaporated todryness using a rotary evaporator. The residue was dissolved in ethylacetate, washed with water and brine, then was dried over MgSO₄,filtered, and evaporated to provide the product.

Other amino-linker alcohols may be prepared according to this generalmethod.

Example 11 Preparation of ^(t)BOC-Amino-Linker Alcohols

A solution of the amino-linker alcohol of Example 10 (R=phenyl; 1.0mmol) in 2 mL of THF was treated with di-tert-butyl dicarbonate (1.5mmol) for 1 hour, and then evaporated to dryness. The residue wasdissolved in ethyl acetate, washed with water and brine, then was driedover MgSO₄, filtered, and evaporated to provide the product.Chromatography on silica gel using a gradient of ethyl acetate in hexaneprovided the purified product.

Other ^(t)BOC-amino-linker alcohols may be produced according to thesame general method.

Example 12 Preparation of 4-(N,N-Diethylcarboxamido)aniline

(1) N,N-diethyl 4-nitrobenzamide: Diethylamine (5.6 mL) was added to anice-cold solution of 4-nitrobenzoyl chloride (5.0 g) in 100 mL of DCM.After 1 h, the mixture was washed successively with water, sat. aq.NaHCO₃, and brine, then dried over MgSO₄, filtered, and evaporated toprovide a colorless liquid that crystallized on standing.Recrystallization from ethyl acetate/hexane provided the product as paleyellow crystals (4.6 g).

(2) 4-(N,N-diethylcarboxamido)aniline: A mixture of N,N-diethyl4-nitrobenzamide (4.44 g) and 10% palladium on carbon (0.2 g) in 100 mLof methanol was treated with ammonium formate (4.0 g) for 2 h at ambienttemperature. The mixture was filtered through Celite and concentrated.The residue was redissolved in DCM, washed successively with 0.5 MNa₂CO₃, water, and brine, then dried over MgSO₄, filtered, andevaporated to provide a crystalline material. Recrystallization fromethyl acetate/hexane provided the product aniline.

Also prepared according to the same procedure was4-(morpholinocarbonyl)aniline by replacing diethylamine with morpholine.

Example 13 Preparation of Azidocarbamates

The crude chloroformate prepared from 2.5 mmol of azidoalcohol accordingto the procedure of Example 5 was dissolved in 20 mL of THF, and theaniline (2.5 mmol) and triethylamine (0.7 mL, 5.0 mmol) were added.After 1 h, the mixture was diluted with ethyl acetate, washedsuccessively with 1 N HCl, water, sat. NaHCO₃, and brine, then driedover MgSO₄, filtered, and evaporated. The residue was chromatographed onsilica gel using ethyl acetate/hexane to provide the product carbamate.

Compounds prepared according to this method include:

-   O-[1-(phenylsulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl    carbamate;-   O-[1-(morpholinosulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl    carbamate;-   O-[1-(methanesulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl    carbamate;-   O-[1-(phenylsulfonyl)-7-azido-2-heptyl]-N-[4-(morpholinocarboxamido)phenyl    carbamate; and-   O-[1-(phenylsulfonyl)-7-azido-2-heptyl]-N-[4-(morpholinosulfonyl)phenyl    carbamate.

Example 14 Preparation of N-Chloromethyl Carbamates

A mixture of the azidocarbamate of Example 13 (1.0 mmol),paraformaldehyde (45 mg), chlorotrimethylsilane (1 mL), and THF (1 mL)in a sealed 20 mL vial was heated in a 55° C. bath for 17 h. Aftercooling to ambient temperature, the vial was opened and the mixture wasconcentrated on a rotary evaporator to a thick oil, which was taken upin ethyl acetate and reconcentrated. The residue was dissolved in 2:1ethyl acetate/hexane, filtered, and concentrated to provide theN-chloromethyl carbamate, which was used without further purification.

Compounds prepared according to this method include:

-   O-[1-(phenylsulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N-chloromethyl    carbamate;-   O-[1-(morpholinosulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N-chloromethyl    carbamate; and-   O-[1-(methanesulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N-chloromethyl    carbamate.

Example 15 Preparation of N-Alkoxymethyl Carbamates

The N-chloromethyl carbamate of Example 14 (0.4 mmol) was dissolved in 5mL of dry methanol. After 1 h, the mixture is evaporated to dryness, andthe residue was chromatographed on silica gel (ethyl acetate/hexanes) toprovide the product.

Compounds prepared according to this method include:

-   O-[1-(phenylsulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N-methoxymethyl    carbamate;-   O-[1-(morpholinosulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N-methoxymethyl    carbamate; and-   O-[1-(methanesulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N-methoxymethyl    carbamate.

Example 16 7-(Tert-Butoxycarbonylamino)-2-(R¹-Sulfonyl)-1-Heptanol

p-Toluenesulfonyl chloride (1 mmol) is added to a solution of6-azido-1-hexanol (Example 1, 1 mmol) in pyridine (2 mL) cooled on ice.After 30 min, the mixture is allowed to warm to ambient temperature andtreated with R¹—SH (1 mmol) for an additional 1 hr. The mixture isdiluted with ethyl acetate, washed sequentially with water, 1 N HCl,water, sat. aq. NaHCO₃, and brine, then dried over MgSO₄, filtered, andevaporated. The crude thioether is dissolved in ethyl acetate andtreated excess peracetic acid to prepare the sulfone. After standardaqueous workup, the sulfone is purified by chromatography on silica gel.A mixture of the sulfone, ethyl formate, and 2 equivalents of sodiumhydride in DMF is warmed to 50° C. to provide an intermediate aldehyde,which is treated with sodium borohydride in methanol to produce theproduct alcohol.

Example 17

A solution of an amino-thiol heterobifunctional PEG in THF is treatedwith excess di-tert-butyl dicarbonate until the reaction is complete,and the di-BOC product is isolated by chromatography. The thiocarbonateis cleaved by treatment with one equivalent of NaOMe in methanol, and2-bromoethanol is added to form the hydroxyethyl thioether, which isoxidized with peracetic acid to form the product.

These compounds may be prepared by a method analogous to that describedfor methoxy-PEG-hydroxyethyl sulfone (Morpurgo, et al., BioconjugateChemistry (1996) 7:363-368, incorporated herein by reference). Forexample, a solution of 11-azido-3,6,9-trioxaundecan-1-ol (x=3) (3 mmol)in toluene is dried by azeotropic distillation. After dissolution inCH₂Cl₂, methanesulfonyl chloride is added followed by triethylamine toform the mesylate. A solution of the mesylate in water is treated with2-mercaptoethanol and 2 N NaOH to form the hydroxyethyl sulfide. Thesulfide is subsequently oxidized to the sulfone, for example usinghydrogen peroxide in the presence of a tungstic acid catalyst oralternatively using peracetic acid. The hydroxyethyl sulfone is thenactivated as the succinimidyl carbonate according to the methodsdescribed in the examples above.

Example 19

Example 20 Preparation of Crosslinkers of Formula (1)

A solution of 7-azido-1-(phenylsulfonyl)-2-hepyl succinimidyl carbonate(119 mg, 0.27 mmol) in 2 mL of acetonitrile was treated with11-azido-3,6,9-trioxaundecan-1-amine (65 mg, 0.30 mmol) for 10 min atambient temperature. After evaporation of the solvent, the residue wasdissolved in 1 mL of CH₂Cl₂ and chromatographed on a 4-g column ofsilica gel using a step gradient of hexane, 3:1 hexane/ethyl acetate,1:1 hexane/ethyl acetate, and 1:2 hexane/ethyl acetate. Theproduct-containing fractions were pooled and evaporated to provide theproduct.

Example 21 Preparation of 4-arm PEG-[DBCO]4

A solution of 40-kDa 4-arm polyethylene glycol with aminopropylend-groups having a pentaerythritol core (NOF America, PTE400PA) (500mg, 12.5 μmol), triethylamine (20 μL), and6-aza-5,9-dioxo-9-(1,2-didehydrodibenzo[b,f]azocin-5(6H)-yl)nonanoicacid succinimidyl ester (“DBCO-NHS”, Click Chemistry Tools, Macon, GA)(36 mg, 75 μmol) in 5 mL of THF was stirred for 24 h at ambienttemperature. The product was precipitated by addition of the reactionmixture to 50 mL of methyl tert-butyl ether (MTBE). The precipitate wascollected by vacuum filtration and dried under vacuum to provide 510 mgof product.

Example 22 Hydrogel Formation

A solution of 4.5 mg of 4-arm PEG-[DBCO]₄ (Example 21) in 100 μL of 10mM acetate buffer, pH 5, was treated with 5.0 L of a 40 mg/mL solutionof the diazide crosslinker of Example 20. The solution rapidly set toprovide an elastic hydrogel.

Similarly, a solution of 4.5 mg of 4-arm PEG-[DBCO]₄ (Example 21) in 100μL of 10 mM acetate buffer, pH 5, was treated with 2.5 L of a 40 mg/mLsolution of the diazide crosslinker of Example 20. The solution gelledto produce a viscous hydrogel.

Example 23 Preparation of Multivalent PEG-(Linker-Azide)_(x)Crosslinking Reagents of Formula (2)

The preparation of multivalent PEG-(linker-azide)_(x) crosslinkingreagents is exemplified by the preparation of a compound of formula (2)wherein m=0, n=approximately 100, s=0, t=4, W═O(C═O)NH, Q=C(CH₂)₄,R¹=PhSO₂, R²═H, one R⁵═H and the other R⁵═(CH₂)₅N₃. Other compounds offormula (2) were prepared using the same method by substitution of theappropriate azide-linker-succinimidyl carbonate of Example 7. Asnecessary, analogous azide-linker-succinimidyl carbonates of otherExamples may also be used.

Thus, a solution of 25 μmol of the azido-linker-succinimidyl carbonate(Example 7) in 1 mL of ACN was added to a mix of 5 μmol (100 mg) of20-kDa 4-arm PEG-amine hydrochloride (pentaerythritol core, JenKemTechnologies) in 1 mL of water and 40 μL of 1.0 M NaHCO₃ (40 μmol).After 1 hr at ambient temperature the solution was dialyzed (12-14 kMWCO) against 1 L of 50% methanol followed by 1 L of methanol. Afterevaporation, the residue (109 mg) was dissolved in 2.12 mL ofsterile-filtered 10 mM NaOAc, pH 5.0, and stored frozen at −20° C. Theazide concentration determined by reaction with DBCO-acid was 9.5 mM.

Example 24 Preparation of Multivalent PEG-(Cyclooctynes)_(x)

PEG_(20 Kda)-(Dbco)₄:

A 60 mM solution of freshly chromatographed DBCO-NHS (Click ChemistryTools) in acetonitrile (0.5 mL, 30 μmol, 1.5 eq) was added to a solutionof 20 kDa 4-arm PEG-amine hydrochloride (pentaerythritol core, JenKemTechnologies; 100 mg, mol), and diisopropylethylamine (0.010 mL, 57μmol) in acetonitrile (1 mL). After stirring 2 h at ambient temperature,the mixture was evaporated to dryness under reduced pressure. Theresidue was dissolved in 50% aqueous methanol (4 mL) and dialyzedagainst 50% aqueous methanol followed by methanol. After evaporation,the residue (100 mg) was dissolved in water to give a 50 mg/mL stock (10mM DBCO by spectrophotometric assay), which was stored frozen at −20° C.

PEG_(40 Kda)-(Dbco)₈:

One mL of 40 mM solution (40 μmol) of DBCO-NHS in THF was added to asolution of 168 mg (4.2 μmol) of 40-kDa 8-arm PEG-amine hydrochloride(tripentaerythritol core, JenKem Technologies) and 12.9 μLdiisopropylethylamine (74 μmol) in 0.6 mL of ACN, and the mixture waskept at ambient temperature overnight. The reaction mixture was dialyzedagainst 2 L of 50% methanol followed by 1 L of methanol. Afterevaporation, the residue (149 mg) was dissolved in 1.49 mL water andstored frozen at −20° C. The DBCO concentration determinedspectrophotometrically was 16 mM.

PEG_(40 kDa)(BCN)₈:

A solution of 200 mg of 40 kDa 8-arm PEG-amine*HCl (JenKem Technologies;40 μmol NH₂), 20 mg of BCN p-nitrophenyl carbonate (SynAffix; 63 μmol),and 20 μL of N,N-diisopropylethylamine (115 μmol) in 2 mL of DMF wasstirred 16 h at ambient temperature. After quenching with 0.5 mL of 100mM taurine in 0.1 M KPi, pH 7.5, for 1 h, the mixture was dialyzedsequentially against water, 1:1 methanol/water, and methanol using a 12kDa membrane. After evaporation, the residue was dissolved in 2 mL ofTHF and precipitated with 10 mL of methyl ^(t)butyl ether. The productwas collected and dried (190 mg).

Example 25 Preparation of BODIPY-Azide Erosion Probe

A 100 mM solution of 11-azido-3,6,9-trioxaundecan-1-amine inacetonitrile (13 μL, 13 μmol) was added to a 12.8 mM solution of BODIPYTMR-X SE (Invitrogen) in DMSO (100 μL, 1.28 μmol). After 30 min atambient temperature, the mixture was diluted to 2 mL with 0.1 M KPi, pH7.4, and loaded on a 500 mg C18 BondElut™ extraction column (Varian).The column was washed successively with 5 mL portions of water and 20%ACN/water, then eluted with 50% ACN/water and concentrated to dryness.The residue was dissolved in 1.0 mL of ACN and the concentration (1.0mM) was determined using ε₅₄₄ nm=60,000 M⁻¹ cm⁻¹.

Example 26 Preparation of Fluorescein-Azide Erosion Probe

A 10 mg/mL solution of 5-(aminoacetamido)fluorescein (Invitrogen) in DMF(100 μL) was mixed with a 25 mM solution of 6-azidohexyloxy succinimidylcarbonate (100 μL) for 1 h to provide a 12.5 mM solution of thefluorescein-azide erosion probe.

Example 27 Preparation of Hydrogels Using Multivalent CrosslinkingReagents of Formula (2)

For preparation of 4×4 hydrogels, a 50 mg/mL solution ofPEG_(20 kDa)(DBCO)₄ (Example 24; 250 μL, 2.5 μmol DBCO end-groups) inwater was mixed with 25 μL of a 10 mM solution of the fluorescein-azideerosion probe in DMF (Example 26; 0.25 μmol azide) and kept 30 min atambient temperature. Fifty L aliquots (0.42 μmol DBCO) were mixed with28 μL of 10 mM NaOAc, pH 5.0, followed by 42 μL of 50 mg/mLPEG_(20 kDa)(linker-azide)₄ (Example 23; 0.42 μmol azide). Componentswere mixed by vortexing, centrifuged briefly to remove any air bubbles,and quickly pipetted into 64 μL (9×1 mm) circular rubber perfusionchambers (Grace Bio-Labs) mounted on a silanized glass microscope slide,and allowed to cure overnight.

Preparation of 4×8 hydrogels followed the same method, using solutionsof PEG_(40 kDa)(DBCO)₈ or PEG_(40 kDa)(BCN)₈ (Example 24) in place ofPEG_(20 kDa)(DBCO)₄ and adjusting the proportions of 8-armed cyclooctyneand 4-armed linker-azide monomers so as to provide gels having thedesired total wt % PEG and degree of crosslinking.

Example 28 Measurement of Reverse Gelation Times

Gel discs (Example 27) were suspended in buffer at 37° C., and OD₄₉₃ inthe solution was periodically measured to monitor fluoresceinsolubilization. The reverse gelation times (tRGEL) were those times whengels were completely solubilized. The pH dependence of the degelationtime was determined using 4×4 gels (5% total PEG by weight) preparedfrom PEG_(20 kDa)(DBCO)₄ crosslinked using a compound of formula (2)wherein m=0, n=approximately 100, s=0, t=4, W═O(C═O)NH, Q=C(CH₂)₄,R¹=(4-chlorophenyl)SO₂, R²═H, one R⁵═H and the other R⁵═(CH₂)₅N₃. Thegel discs were suspended in buffers from pH 7.8-9.0. Degelation curvesare shown in FIG. 5, with measured times at pH 7.8=20.9 h, pH 8.1=10.9h, pH 8.4=5.6 h, pH 8.7=2.8 h, and pH 9.0=1.5 h. As shown in FIG. 6, thedegelation time varies linearly with pH, increasing 10-fold for eachdrop of 1 pH unit.

The effect of the linker modulator R¹ on degelation time was determinedby preparing hydrogel discs from PEG_(20 kDa)(DBCO)₄ crosslinked usingcompounds of formula (2) wherein m=0, n=approximately 100, s=0, t=4,W═O(C═O)NH, Q=C(CH₂)₄, R²═H, one R⁵═H and the other R⁵═(CH₂)₅N₃, andwherein R¹ was either (4-chlorophenyl)SO₂, phenyl-SO₂, morpholino-SO₂,or CN. A control gel was prepared having no modulator (R¹R²CH isabsent). Degelation curves of the discs suspended in KPi, pH 7.4, 37°C., are shown in FIG. 3. As shown in FIG. 4, there is a linearcorrelation between the half-life of linker cleavage as determined byrelease of 5-(aminoacetamido)fluorescein (see Santi, et al., Proc. Nat.Acad. Sci. USA (2012) 109:6211-6216), incorporated herein by reference,and the degelation time of the corresponding hydrogel.

Example 29 Controlled Drug Release from Hydrogels

Hydrogels were prepared from PEG_(40 kDa)-(DBCO)₈ wherein a fraction ofthe cyclooctynes were first reacted with a small amount of azide erosionprobe and with an azide-linker-drug of formula (3) wherein the linkercomprised a modulating group R¹, then crosslinked using a compound offormula (2) wherein m=0, n=approximately 100, s=0, t=4, W═O(C═O)NH,Q=C(CH₂)₄, R²═H, one R⁵═H and the other R⁵═(CH₂)₅N₃, and wherein R¹ waseither (4-chlorophenyl)SO₂, phenyl-SO₂, morpholino-SO₂, or CN. Themodulating groups of the azide-linker-drug of Formula (3) and thecompound of formula (2) were chosen such that release of drug wouldoccur more rapidly than erosion and subsequent degelation of thehydrogel.

In one example, gels were prepared using 5-(acetamido)fluorescein (AAF)as a drug surrogate. The modulating R¹ groups in Formula (3) were variedas noted below. Thus a solution (99.6 μL) containing 50 μL of 100 mg/mLPEG_(40 kDa)-(DBCO)₈ (1.0 μmol DBCO end groups) in water was mixed with6.2 μL of 12.5 mM of azide-linker-AAF (0.078 μmol) in 1:1DMF:acetonitrile (where the linker comprised one of various modulators),15 μL of 1.0 mM BODIPY-azide (0.015 μmol) in acetonitrile as an erosionprobe, 20 μL of 20 mM O-(2-azidoethyl)heptaethylene glycol (0.40 μmol)in water to cap excess cyclooctynes, and 8.4 μL water. After 10 min atambient temperature, the solution containing 0.5 μmol uncommitted DBCOgroups was mixed with 50 μL of a 50 mg/mL solution of the compound offormula (2) wherein R¹═CH₃—SO₂ (0.5 μmol azide groups) in 10 mM NaOAc,pH 5.0.

Duplicate cast gels were suspended in 0.1 M HEPES, pH 7.4, at 37° C.,and OD₄₉₃ for fluorescein and OD₅₄₆ for BODIPY in the solution wasperiodically measured. The release times for fluorescein where R¹ inFormula 3 is of various groups was measured as shown in FIG. 7. Thereverse gelation time, as determined by complete solubilization of theBODIPY erosion probe, was 630±39 (S.D.) hr (n=8). Solubilization offluorescein followed the first-order rate law[F]_(t)/F_(tot)=exp(−k_(obsd)t) and gave apparent k_(obsd)±S.E. for thetotal released fluorescein of 0.021±0.00014 hr⁻¹ for R¹=4-ClPh-S O₂—,0.011±0.00031 hr⁻¹ for R¹=Ph-SO₂—, 0.0053±0.00022 hr⁻¹ forR¹=4-MeO-Ph-SO₂—, and 0.0033±0.00010 hr⁻¹ for R¹=MeSO₂—. The rate datawere converted to plots for the fluorescein released directly from thegel using Eq. S6 (Example 30).

The pH-dependence of drug release was determined by observing AAFrelease from the above gels prepared using R¹=(4-chlorophenyl)SO₂between pH 7.4 and 9.0. As shown in FIGS. 8 and 9, the rate of drugrelease increases with increasing pH.

Example 30 Modeling of Drug Release and Gel Erosion

Drug release and gel degradation occurs as follows, with the finalproducts being the free drug and gel monomers:(Gel)-(Drug)_(n)→Drug+EP-gel fragment-Drug→Drug+EP-monomers

The drug or drug surrogate released into solution may emanate directlyfrom L1 cleavage from the gel, or from solubilized fragments that arisefrom gel erosion via cleavages of L2. To distinguish the drug releasedfrom the intact gel vs. solubilized gel fragments, it is necessary todetermine the distribution of drug-bearing nodes between the intact geland solution at time t. In the present study, we used a modification ofa reported approach to monitor and model gel degradation (2). Theappearance of an erosion probe EP permanently attached to nodes of thegel allows calculation of the fraction of nodes in solution asEP(t)/E_(∞); the concentration of drug originally present on thesesolubilized nodes, D_(s)(t), is thus given by Eq. S1.D _(s)(t)=D _(∞)*EP(t)/EP_(∞) or (D _(∞)/EP_(∞))*EP(t)  [S1]

The drug released from the intact gel at time t, D_(g)(t), is thedifference between the total drug released, D(t), and the drug eithercontained in or released from solubilized gel fragments D_(s)(t), as inEq. S2.D _(g)(t)=D(t)−D _(s)(t)=D(t)−(D _(∞)/EP_(∞))*EP(t)  [S2]

Calculation of the first-order rate of drug release from intact gelnodes is not straightforward from measuring D(t) due to the changingquantity of gel from erosion, but can be calculated based on thefraction of drug remaining on intact gel. Based on released erosionprobe EP(t), the fraction of gel remaining is 1−EP(t)/EP_(∞). The amountof drug originally carried by this amount of gel is thus given byD_(∞)*(1−EP(t)/EP_(∞)). As the drug remaining on the intact gel isD_(∞)−D(t), the fraction of drug remaining on intact gel, D_(f,gel)(t)is given as Eqs. S3-S4.D _(f,gel)(t)=[D _(∞) −D(t)]/[D _(∞)*(1−EP(t)/EP_(∞))]  [S3]=[1−D(t)/D _(∞)]/[1−EP(t)/EP_(∞)]  [S4]

For a first order release of drug from the gel, D_(f,gel)(t) will showan exponential decay having a rate constant k_(L1) that describes therate of drug release from intact gel, Eq. S5. Merging Eq. S4 and S5provides S6 which can be used to experimentally estimate the rate ofdrug release directly from intact gel.D _(f,gel)(t)=e ^(−k) ^(L1) ^(t)  [S5]D _(f,gel)(t)=[1−D(t)/D _(∞)]/[1−EP(t)/EP_(∞)]=e ^(−k) ^(L1) ^(t)  [S6]

The amount of drug released by the gel over time depends on the rate ofrelease, k_(L1), together with the erosion rate of the gel. If thesolubilization of the erosion probe can be approximated by a first orderprocess between times t=0 and t₁ with rate k_(sol), the quantity of drugreleased from the gel during that time can be approximated as Eq. S7.D _(g)(t ₁)=D _(∞)*(k _(L1)/(k _(sol)))*[1−e ^(−(k) ^(sol) ^()tl)]  [S7]

If the drug remaining on the intact gel is negligible at time t₁, thenthe total fraction of drug directly released from the gel is given inEq. S8D _(g)(t ₁)/D _(∞) =k _(L1) /k _(sol) =t _(1/2,sol) /t _(1/2.L1)  [S8].

Example 31 Effect of Crosslink Density on Degelation Time

As detailed in Table 1, a mixture of 100 mg/mL PEG_(40 kDa)-(BCN)₈ (20mM BCN end-groups) in water was combined with appropriate amounts of 10mM fluorescein-azide and the compound of Formula (2) wherein m=0,n=approximately 100, s=0, t=4, W═O(C═O)NH, Q=C(CH₂)₄, R²═H, one R⁵═H andthe other R⁵═(CH₂)₅N₃, and R¹=(4-chlorophenyl)SO₂ (10 mM azide) in waterand 50 mM O-azidoethyl-heptaethylene glycol in water to prepare 4% PEGhydrogels having 4, 5, 6, 7, or 7.8 crosslinks per 8-arm PEG monomer.Cast gels were placed in 1 mL of 0.1 M borate, pH 9.2, and kept at 37°C. Dissolution of the gels was monitored by appearance of OD₄₉₃ in thesupernatant.

TABLE 1 Preparation and degelation times of gels with varyingcrosslinking densities. Crosslinks/ 8-arm PEG 4 5 6 7 7.8 PEG-(BCN)₈ 40μL 36.9 μL 34.3 μL 32.0 μL 30.4 μL Fluorescein- 1.5 μL 1.5 μL 1.5 μL 1.5μL 1.5 μL azide Cap-azide 7.7 μL 5.2 μL 3.1 μL 1.3 μL 0 μL PEG-(L2-N₃)₄40 μL 46.2 μL 51.4 μL 56.0 μL 59.2 μL Water 60.8 μL 60.2 μL 59.7 μL 59.2μL 58.9 μL Degelation 0.62 h 0.77 h 0.83 h 0.88 h 0.97 h time (pH 9.2)Degelation 37 h 46 h 50 h 53 h 58 h time (pH 7.4)

Gels dissolved at pH 9.2 with degelation times as indicated in Table 1,with the degelation time at pH 7.4 calculated as (degelation time at pH9.2)*10^((9.2−7.4)) as determined in Example 28. As expected, degelationtime increased with increasing number of crosslinks to each 8-armmonomer.

Example 32 Preparation of an Exenatide-Releasing Degradable Hydrogel

Exenatide linked at the α-terminus to an azide-linker having R¹=MeSO₂—as modulator was synthesized by solid-phase peptide synthesis at AnaSpec(Fremont, Calif.) as previously described (Santi, et al., Proc. Nat.Acad. Sci. USA (2012) 109:6211-6216), resulting in a compounds offormula (3) wherein R¹=MeSO₂, R²═H, m=0, one R⁵═H and the otherR⁵═(CH₂)₅N₃, Y═NH, and D=exenatide linked via the N-terminal aminogroup. Azide-linker-exenatide (1.2 mg, 270 nmol) in 30 μL of 1.0 Mphosphate, pH 7.8, and 8-arm PEG_(40 kD)-(BCN)₈ (Example 24; 5 mg; 50μL, 1000 nmol BCN groups) in 50 μL of water was kept for 1 hr at ambienttemperature, then 20 μL of a 1 mM BODIPY-azide (20 nmol) in ACN aserosion probe and a crosslinker of formula (2) wherein m=0,n=approximately 100, s=0, t=4, W═O(C═O)NH, Q=C(CH₂)₄, R²═H, one R⁵═H andthe other R⁵═(CH₂)₅N₃, and R¹═CN (3.55 mg; 710 nmol N₃ groups; Example23) in 71 water was added. The gels were allowed to cure overnight, thenstored in 1 mL of PBS, pH 7.4, at 4° C.

Example 33 Release of Exenatide from an Exenatide-Releasing DegradableHydrogel

A gel disc (Example 32) was placed in 1.0 mL of 0.1 M borate buffer, pH8.8, and kept at 37° C. Solubilization of exenatide (either as freepeptide or as solubilized gel-exenatide fragments) and gel erosion weremonitored at 280 nm and 544 nm, respectively, by periodic sampling ofthe supernatant. These results are shown in FIG. 10. Release wascalculated as solubilization adjusted for gel erosion. Exenatidesolubilization was a first-order process with t_(1/2)=20.7 h at pH 8.8which, assuming the reaction is first order in hydroxide ion,corresponds to a half-life of 520 h (21 days) at pH 7.4; a t_(1/2) of23.6 h at pH 8.8, corresponding to 593 h (24.7 d) at pH 7.4 wascalculated for the drug directly released from the gel (Example 30),which accounted for ˜87.8% of the total exenatide. Reverse gelation wasobserved at 172 h at pH 8.8, corresponding to approximately 180 days atpH 7.4.

Example 34 Diffusion of Encapsulated Proteins from Hydrogels

Stock solutions of ˜90 OD₂₈₀/mL myoglobin (17.7 kDa), carbonic anhydrase(29.0 kDa), and BSA (66.4 kDa) were prepared in 0.1 M KPi, pH 7.4. PEGhydrogels (4%) were prepared by adding 100 mg/mLPEG_(20 kDa)-(NHCO₂(CH₂)₆N₃)₄ (50 uL) to a mixture of 100 mg/mL 20 kDaPEG-(DBCO)₄ (50 μL), protein stock (50 μL), and 10×-PBS (100 μL). Castgels were suspended in 2 mL of 0.1 M KPi, pH 7.4, at 37° C., and OD₂₈₀in the solution was periodically measured. The t_(1/2) values forrelease into solution were −20 min for myoglobin 24 min for carbonicanhydrase and 150 min for BSA.

Example 35 Preparation of Derivatized Hyaluronic Acids

Sodium Hyaluronate of mw=1.6×10⁶ (Lifecore Biomedical; 10.4 mg, 0.0275mmol carboxylate) was treated with a solution of4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM; 30.4 mg, 0.110 mmol, 4 equiv) in 1.05 mL of 0.1 M MES buffer, pH5.5. The resulting mixture was shaken vigorously for 15 min to dissolve.A solution of DBCO-PEG₄-NH₂ (Click Chemistry Tools; 0.113 mL of 24.3 mMin 2:1 ACN:MeOH, 0.00275 mmol, 0.1 equiv) in 0.3 mL of MES buffer wasadded. The resulting mixture was allowed to stand for 24 h then analyzedfor the consumption of free amine by TNBS assay at 3.5 and 24 h asfollows: 0.05 mL of the reaction mixture was diluted to 1 ml in 0.075 Mborate buffer (pH 9.34) containing 0.004% w/v2,4,6-trinitrobenzesulfonic acid and 25% methanol. The absorbance of thereaction at 420 nm was followed until stable (˜1 h). Reactionscontaining amounts of DMTMM, hyaluronic acid, or DBCO-PEG₄-NH₂ were usedas controls. Upon completion, the reaction mixture was diluted with 8 mLof water and dialyzed (12000-14000 MWCO) five times against water thenonce against methanol. The dialyzed product was concentrated to drynessunder reduced pressure and desiccated under hard vacuum over P₂O₅ togive DBCO-hyaluronic acid (11 mg, ˜0.029 mmol disaccharide) as a cleardry glassy solid. This material was dissolved in 3 mL of water to giveslightly greasy very viscous solution containing 0.276 mM DBCO (based onε₃₀₉=13,448 M⁻¹cm⁻¹. This corresponds to a degree of substitution of2.9% (5.3% based on amine consumed in TNBS assay). Hyaluronic acids ofdifferent molecular weights may be derivatized with cyclooctynereagents, such as DIFO or BCN, according to this method.

Amine-derivatized hyaluronic acids were prepared according to thefollowing method. To a solution of sodium hyaluronate of MW=76,000(Lifecore Biomedical; 154 mg, 0.385 mmol disaccharide/carboxylate) inwater (4 mL) was added 1,3-diaminopropane (0.973 mL, 856 mg, 11.6 mmol,30 equiv). The pH of the resulting solution was adjusted to 7.0 with 6 NHCl (final volume ˜7 mL) then solid N-hydroxysuccinimide was added (177mg, 1.54 mmol, 4 equiv), followed by solid1-(3-dimethylamino)propyl-3-ethylcarbodiimide HCl salt (294 mg, 1.54mmol, 4 equiv). The reaction became acidic as it progressed (pH 5.3after 10 min). Every 10 min the pH was adjusted back to 7.2 until stable(˜1 h). After stirring for 18 h the mixture was dialyzed (12-14 k MWCO)against PBS, 5% NaCl, twice against water, then against methanol. Themixture was concentrated to dryness to give 85 mg ofpropylamino-hyaluronic acid as a white solid. An aliquot of thismaterial (7.4 mg, ˜0.019 mmol disaccharide) was dissolved in water (0.5mL) to give a solution of ˜38 mM disaccharide. This solution (0.025 mL)was assessed for free amine content by TNBS assay by incubating in pH9.36 borate buffer (1 mL) containing 0.02% w/v TNBS. The absorbance at420 nm was monitored until stable (˜1 h). The assay indicated a degreeof substitution of 7%.

To a solution of mw=1.6×10⁶ 7% DS propylamino HA (0.5 mL of 0.64 mM NH₂,320 nmol NH₂) in water was added 0.1 mL of 100 mM PBS, followed by asolution of DBCO-PEG₄-NHS ester (Click Chemistry Tools; 0.0308 mL of 25mM as determined by 8309=13,449 M⁻¹cm⁻¹, 770 nmol, 2.4 equiv) inmethanol. The resulting mixture was allowed to sit for 4 hours. TNBSassay indicated loss of 81% of the available amines on the derivatizedhyaluronic acid. A parallel reaction using 1.2 equivalent ofDBCO-PEG₄-NHS ester resulted in consumption of 64% of the availableamines. For purification, the two reactions were combined and dialyzed(12-14 k MWCO) against PBS, then 5% w/v NaCl, then twice against water,then once against methanol. The dialysis mixture was concentrated todryness to give 2.6 mg of a white glassy solid. This material wasdissolved in 1 mL of water to give a solution of 6.5 mM disaccharide and0.31 mM DBCO based on ε₃₀₉=13,448 M⁻¹cm⁻¹, corresponding to a DBCOsubstitution of 4.8% and a yield of acylation of 71%.

Example 36 Preparation of Hyaluronic Acid Hydrogels

Hyaluronic acid hydrogels are prepared by crosslinkingcyclooctyne-derivatized hyaluronic acid (Example 35) with diazidecrosslinkers of formula (1) wherein m=0, X═O—CO—NH—(CH₂CH₂O)₃CH₂CH₂N₃,R¹=PhSO₂, R²═H, one R⁵═H and the other R⁵═(CH₂)₅N₃. Gel formation istypically performed in water or buffered water using a 2:1 molar ratioof cyclooctyne to diazide crosslinker, optionally in the presence of asolution of protein or small molecule to be encapsulated.

To study diffusion of proteins from the hyaluronic acid hydrogel matrix,a stable hydrogel was prepared by mixing a solution (0.065 mL) ofDBCO-HA (Example 35), 6.6% DS DBCO, 3.9 mM DBCO) in water with asolution of diazido-PEG of either MW=2000 or 5000 (0.005 mL of 25 mM,0.5 equiv/DBCO). This hydrogel master mix 0.07 mL was immediately mixedwith a protein or small molecule substrate solution (0.01 mL) forencapsulation in the bottom of a standard plastic 2.5 mL cuvette. Thehalf-lives for diffusion from the gels are given in Table 2 below:

TABLE 2 Lys carbonic Substrate (DNP) myoglobin anhydrase BSA IgG Mw 31218,000 29,000 66,000 150,000 t_(1/2) (2K gel) 0.96 h 3.98 h 3.66 h 4.26h 5.71 h t_(1/2) (5K gel) 1.25 h 3.14 h 3.36 h 3.65 h 3.32 h

Alternatively, drugs may be releasably linked to the hyaluronic acidprior to gel formation by reaction of a subset of the availablecyclooctynes with azide-linker-drug as described in Example 29 andExample 32 above. In this case, the amount of diazide crosslinker usedfor gel formation is calculated based on the available cyclooctynesremaining after drug attachment. Attachment of5-(aminoacetamido)fluorescein via a linker with RD=(4-chlorophenyl)SO₂provided a hyaluronic acid hydrogel that released AAF with t_(1/2)=49 hat pH 7.4, 37° C.

Example 37 Method for Preparing Hydrogels with ControlledStoichiometries

As depicted in FIG. 11, commercially available S-t-Butylthio-cysteine (HCys(^(t)BuS)) is acylated with a cyclooctyne succinimidyl ester (e.g.DBCO-HSE or BCN-HSE) to give CO-Cys(^(t)BuS)OH (A′=COOH; B=cyclooctyne;C=^(t)BuS). A 4-arm amino PEG (A=NH₂) is acylated (e.g., using acarbodiimide) with this CO-Cys(tBuS)OH to give theCO/tBuS-functionalized PEG. An azido-linker(R¹)-drug is coupled to thecyclooctyne residues, then the tBuS group is removed, for example usinga thiol such as dithiothreitol or with a phosphine such as TCEP, and thethiol-derivatized PEG is purified of small thiols (for example, usingdialysis or gel filtration chromatography) and reacted with acyclooctyne-maleimide, cyclooctyne-haloacetamide, orcyclooctyne-vinylsulfonamide to introduce exactly 4 cyclooctyne gelationsites per molecule. This intermediate is then crosslinked to form ahydrogel using a compound of formula (1) or (2) wherein the reactivefunctional groups are azide. Alternatively, the thiol-derivatized PEG(prior to reaction with cyclooctyne-maleimide) could also be polymerizedwith a compound of formula (1) or (2) wherein the reactive functionalgroup is a Michael acceptor or alkylating agent such as maleimide, vinylsulfone, vinyl sulfonamide, acrylate, acrylamide, haloacetate, orhaloacetamide. Orthogonally protected adapters other thanS-t-Butylthio-cysteine may similarly be used, for example suitablyprotected lysines, aspartates, or glutamates or synthetic adapters notbased on amino-acids.

The invention claimed is:
 1. A method to prepare a drug-releasingdegradable hydrogel, which method comprises: (a) providing a firstmulti-armed polyethylene glycol (PEG) polymer wherein each arm isterminated by a group comprising orthogonal first and second functionalgroups, which first orthogonal functional group is different from saidsecond orthogonal functional group, and wherein the group comprisingsaid first orthogonal functional group and said second orthogonalfunctional group comprises cysteine, lysine, or aspartate; (b) reactingsaid first multi-armed PEG polymer of step (a) with a linker-drugconjugate wherein said conjugate comprises a third functional group thatreacts with only the first orthogonal functional group to obtain aderivatized first polymer; and (c) reacting the derivatized firstpolymer obtained in step (b) with a crosslinker coupled to a secondmulti-armed PEG polymer wherein said crosslinker comprises a fourthfunctional group that reacts with only the second orthogonal functionalgroup present on said derivatized first polymer to obtain saiddrug-releasing degradable hydrogel, or (d) reacting said firstmulti-armed PEG polymer of step (a) with a crosslinker coupled to asecond multi-armed PEG polymer wherein said crosslinker comprises afourth functional group that reacts only with the second orthogonalfunctional group of said first multi-armed PEG polymer to obtain acrosslinked polymer, and (e) reacting the crosslinked polymer of step(d) with a linker-drug conjugate, wherein said conjugate comprises athird functional group that reacts with only the first orthogonalfunctional group of said first multi-armed PEG polymer to obtain a saiddrug-releasing degradable hydrogel, wherein the crosslinker of step (c)or step (d) is of formula (1)

wherein m of formula (1) is 0 or 1; X of formula (1) is

wherein T* is O, S, or NR⁶; z is 1-6; Y is (OCH₂CH₂)_(p), wherein p is1-100; and Z² is a functional group coupled to the second multi-armedPEG polymer, and one of R¹, R² and R⁵ of formula (1) comprises thefourth functional group that reacts with only the second orthogonalfunctional group present on said first multi-armed PEG polymer of step(a) or said derivatized first multi-armed PEG polymer of step (b); or Z²is the fourth functional group that reacts with only the secondorthogonal functional group present on said first multi-armed PEGpolymer of step (a) or said derivatized first multi-armed PEG polymer ofstep (b), and one of R¹, R² and R⁵ of formula (1) comprises a functionalgroup coupled to the second multi-armed PEG polymer; with the provisothat: (1) when one of R⁵ of formula (1) comprises the fourth functionalgroup or the functional group coupled to the second multi-armed PEGpolymer, the remaining R⁵ of formula (1) is H, optionally substitutedalkyl, optionally substituted alkenylalkyl, optionally substitutedalkynylalkyl, (OCH₂CH₂)_(p) O-alkyl wherein p=1-1000, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted heteroaryl, or optionally substituted heteroarylalkyl; and(a) R¹ and R² of formula (1) are independently CN, NO₂, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkenyl, optionally substituted alkynyl, COR³, SOR³, SO₂R³,or SR⁴; (b) R¹ and R² of formula (1) may be joined to form a 3-8membered ring; (c) R¹ of formula (1) is CN, NO₂, optionally substitutedaryl, optionally substituted heteroaryl, optionally substituted alkenyl,optionally substituted alkynyl, COR³, SOR³, SO₂R³, or SR⁴; and R² offormula (1) is H, optionally substituted alkyl, optionally substitutedarylalkyl, or optionally substituted heteroarylalkyl; or (d) R² offormula (1) is CN, NO₂, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkenyl, optionallysubstituted alkynyl, COR³, SOR³, SO₂R³, or SR⁴; and R¹ of formula (1) isH, optionally substituted alkyl, optionally substituted arylalkyl, oroptionally substituted heteroarylalkyl; or (2) when R¹ of formula (1)comprises the fourth functional group or the functional group coupled tothe second multi-armed PEG polymer: R² of formula (1) is CN, NO₂,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkenyl, optionally substituted alkynyl, COR³,SOR³, SO₂R³, or SR⁴; and each R¹ of formula (1) is independently H,optionally substituted alkyl, optionally substituted alkenylalkyl,optionally substituted alkynylalkyl, (OCH₂CH₂)_(p) O-alkyl whereinp=1-1000, optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, or optionally substitutedheteroarylalkyl; or (3) when R¹ of formula (1) comprises the fourthfunctional group or the functional group coupled to the secondmulti-armed PEG polymer: R¹ of formula (1) is CN, NO₂, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkenyl, optionally substituted alkynyl, COR³, SOR³, SO₂R³,or SR⁴; and each R⁵ of formula (1) is independently H, optionallysubstituted alkyl, optionally substituted alkenylalkyl, optionallysubstituted alkynylalkyl, (OCH₂CH₂)_(p) O-alkyl wherein p=1-1000,optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, or optionally substitutedheteroarylalkyl; wherein the linker-drug conjugate of step (b) or step(e) is of formula (3)

wherein m of formula (3) is 0 or 1; D of formula (3) is a drug; Y offormula (3) is NH or NBCH₂, wherein B is H, optionally substitutedalkyl, optionally substituted arylalkyl, optionally substitutedheteroaryl, or optionally substituted heteroarylalkyl; either R¹, R² orone of R⁵ of formula (3) comprises the third functional group coupled tothe first orthogonal functional group of the first multi-armed PEGpolymer; with the proviso that: (1) when one of R⁵ of formula (3)comprises the third functional group, the remaining R⁵ of formula (3) isH, optionally substituted alkyl, optionally substituted alkenylalkyl,optionally substituted alkynylalkyl, (OCH₂CH₂)_(p) O-alkyl whereinp=1-1000, optionally substituted aryl, optionally substituted arylalkyl,optionally substituted heteroaryl, or optionally substitutedheteroarylalkyl; and (a) R¹ and R² of formula (3) are independently CN,NO₂, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkenyl, optionally substituted alkynyl, COR³,SOR³, SO₂R³, or SR⁴; (b) R¹ and R² of formula (3) may be joined to forma 3-8 membered ring; (c) R¹ of formula (3) is CN, NO₂, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkenyl, optionally substituted alkynyl, COR³, SOR³, SO₂R³,or SR⁴; and R² of formula (3) is H, optionally substituted alkyl,optionally substituted arylalkyl, or optionally substitutedheteroarylalkyl; or (d) R² of formula (3) is CN, NO₂, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted alkenyl, optionally substituted alkynyl, COR³, SOR³, SO₂R³,or SR⁴; and R¹ of formula (3) is H, optionally substituted alkyl,optionally substituted arylalkyl, or optionally substitutedheteroarylalkyl; or (2) when R¹ of formula (3) comprises the thirdfunctional group: R² of formula (3) is CN, NO₂, optionally substitutedaryl, optionally substituted heteroaryl, optionally substituted alkenyl,optionally substituted alkynyl, COR³, SOR³, SO₂R³, or SR⁴; and each R⁵of formula (3) is independently H, optionally substituted alkyl,optionally substituted alkenylalkyl, optionally substitutedalkynylalkyl, (OCH₂CH₂)_(p) O-alkyl wherein p=1-1000, optionallysubstituted aryl, optionally substituted arylalkyl, optionallysubstituted heteroaryl, or optionally substituted heteroarylalkyl; or(3) when R² of formula (3) comprises the third functional group: R¹ offormula 3) is CN, NO₂, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted alkenyl, optionallysubstituted alkynyl, COR³, SOR³, SO₂R³, or SR⁴; and each R⁵ of formula(3) is independently H, optionally substituted alkyl, optionallysubstituted alkenylalkyl, optionally substituted alkynylalkyl,(OCH₂CH₂)_(p) O-alkyl wherein p=1-1000, optionally substituted aryl,optionally substituted arylalkyl, optionally substituted heteroaryl, oroptionally substituted heteroarylalkyl, wherein each said orthogonalfunctional group independently comprises N₃, NH₂, NH-CO₂ ^(t)Bu, SH,S^(t)Bu, maleimide, CO₂H, CO₂ ^(t)Bu, 1,3-diene, cyclopentadiene, furan,alkyne, cyclooctyne, acrylate, or acrylamide, and wherein when oneorthogonal functional group comprises N₃ the other does not comprisealkyne or cyclooctyne, when one orthogonal functional group comprises SHthe other does not comprise maleimide, acrylate, or acrylamide, when oneorthogonal functional group comprises NH₂ the other does not compriseCO₂H, when one orthogonal functional group comprises 1,3-diene orcyclopentadiene the other does not comprise furan.
 2. The method ofclaim 1, wherein the linker-drug conjugate is degradable by a betaelimination reaction.
 3. The method of claim 1, wherein the crosslinkeris degradable by a beta elimination reaction.
 4. The method of claim 1,wherein in Formula (1) R² is H; and/or one of R⁵ is H and the other is(CH₂)_(n) Z wherein n is 1-6 and Z is said fourth functional group. 5.The method of claim 1, wherein the first and/or second multi-armed PEGpolymer comprises the formula [—(CH₂)_(s)(CH₂CH₂O)_(n)]_(t)Q, wherein nis 10-1000; s is 0-2; t is 2, 4, 8, 16 or 32 and represents the numberof arms of said polymer; and Q is a core group having a valency t. 6.The method of claim 5, wherein t is
 4. 7. The method of claim 5, whereinQ is pentaerythritol, tripentaerythritol, or hexaglycerin.